Suzanne Pfeffer

All Publications

• PathogenicLRRK2mutations cause loss of primary cilia and Neurturin in striatal parvalbumin interneurons. Life science alliance Lin, Y., Jaimon, E., Tonelli, F., Pfeffer, S. R. 2025; 8 (1)

  Abstract

  Parkinson's disease-associated, activating mutations in the LRRK2 kinase block primary cilium formation in cell culture and in specific cell types in the brain. In the striatum that is important for movement control, about half of astrocytes and cholinergic interneurons, but not the predominant medium spiny neurons, lose their primary cilia. Here, we show that mouse and human striatal parvalbumin interneurons that are inhibitory regulators of movement also lose primary cilia. Without cilia, these neurons are not able to respond to Sonic hedgehog signals that normally induce the expression of Patched RNA, and their numbers decrease. In addition, in mouse, glial cell line-derived neurotrophic factor-related Neurturin RNA is significantly decreased. These experiments highlight the importance of parvalbumin neurons in cilium-dependent, neuroprotective signaling pathways and show that LRRK2 activation correlates with decreased Neurturin production, resulting in less neuroprotection for dopamine neurons.

  View details for DOI 10.26508/lsa.202402922

  View details for PubMedID 39537338

• Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease. Proceedings of the National Academy of Sciences of the United States of America Khan, S. S., Jaimon, E., Lin, Y. E., Nikoloff, J., Tonelli, F., Alessi, D. R., Pfeffer, S. R. 2024; 121 (32): e2402206121

  Abstract

  Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.

  View details for DOI 10.1073/pnas.2402206121

  View details for PubMedID 39088390

• Leucine-Rich Repeat Kinases. Annual review of biochemistry Alessi, D. R., Pfeffer, S. R. 2024

  Abstract

  Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65 human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients.

  View details for DOI 10.1146/annurev-biochem-030122-051144

  View details for PubMedID 38621236

• Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B. Science advances Pal, P., Taylor, M., Lam, P. Y., Tonelli, F., Hecht, C. A., Lis, P., Nirujogi, R. S., Phung, T. K., Yeshaw, W. M., Jaimon, E., Fasimoye, R., Dickie, E. A., Wightman, M., Macartney, T., Pfeffer, S. R., Alessi, D. R. 2023; 9 (50): eadj1205

  Abstract

  We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase-mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.

  View details for DOI 10.1126/sciadv.adj1205

  View details for PubMedID 38091401

• Localization of PPM1H phosphatase tunes Parkinson's disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America Yeshaw, W. M., Adhikari, A., Chiang, C. Y., Dhekne, H. S., Wawro, P. S., Pfeffer, S. R. 2023; 120 (44): e2315171120

  Abstract

  PPM1H phosphatase reverses Parkinson's disease-associated, Leucine Rich Repeat Kinase 2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor colocalization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H may maintain active Rab GTPases on the Golgi to carry out their nonciliogenesis-related functions in membrane trafficking.

  View details for DOI 10.1073/pnas.2315171120

  View details for PubMedID 37889931

• Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase. eLife Dhekne, H. S., Tonelli, F., Yeshaw, W. M., Chiang, C. Y., Limouse, C., Jaimon, E., Purlyte, E., Alessi, D. R., Pfeffer, S. R. 2023; 12

  Abstract

  Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. Alphafold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

  View details for DOI 10.7554/eLife.87098

  View details for PubMedID 37874635

• A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation. eLife Vides, E. G., Adhikari, A., Chiang, C. Y., Lis, P., Purlyte, E., Limouse, C., Shumate, J. L., Spinola-Lasso, E., Dhekne, H. S., Alessi, D. R., Pfeffer, S. R. 2022; 11

  Abstract

  Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

  View details for DOI 10.7554/eLife.79771

  View details for PubMedID 36149401

• LRRK2 phosphorylation of Rab GTPases in Parkinson's disease. FEBS letters Pfeffer, S. R. 2022

  Abstract

  Rab GTPases comprise a large family of conserved GTPases that are critical regulators of the secretory and endocytic pathways. The human genome encodes ~65 Rabs that localize to discrete membrane compartments and, when in their GTP-bound state, bind to effector proteins to carry out diverse functions. Activating mutations in LRRK2 kinase cause Parkinson's disease, and a subset of Rab GTPases are important LRRK2 substrates. LRRK2 phosphorylates a conserved threonine residue that is essential for Rab interaction with guanine nucleotide exchange factors, effectors and GDI that recycles Rabs between membrane compartments. This brief review will highlight new findings related to LRRK2-mediated phosphorylation of Rab GTPases and its consequences. Remarkably, Rab phosphorylation flips a switch on Rab effector selection with dominant consequences for cell pathophysiology.

  View details for DOI 10.1002/1873-3468.14492

  View details for PubMedID 36114007

• CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export. The Journal of cell biology Lu, A., Hsieh, F., Sharma, B. R., Vaughn, S. R., Enrich, C., Pfeffer, S. R. 1800; 221 (2)

  Abstract

  We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER-lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.

  View details for DOI 10.1083/jcb.202105060

  View details for PubMedID 34936700

• Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain ELIFE Khan, S. S., Sobu, Y., Dhekne, H. S., Tonelli, F., Berndsen, K., Alessi, D. R., Pfeffer, S. R. 2021; 10

  View details for DOI 10.7554/eLife.67900.sa2

  View details for Web of Science ID 000712035300001

• Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins. Proceedings of the National Academy of Sciences of the United States of America Sobu, Y., Wawro, P. S., Dhekne, H. S., Yeshaw, W. M., Pfeffer, S. R. 2021; 118 (10)

  Abstract

  Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1, and together, these proteins block cilia formation in a variety of cell types, including patient derived iPS cells. We have used live-cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and the loss of cilia seen upon serum readdition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.

  View details for DOI 10.1073/pnas.2005894118

  View details for PubMedID 33653948

• LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade. Life science alliance Dhekne, H. S., Yanatori, I., Vides, E. G., Sobu, Y., Diez, F., Tonelli, F., Pfeffer, S. R. 2021; 4 (5)

  Abstract

  Activating mutations in LRRK2 kinase causes Parkinson's disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va's role in ciliogenesis.

  View details for DOI 10.26508/lsa.202101050

  View details for PubMedID 33727250

• Rab29 Fast Exchange Mutants: Characterization of a Challenging Rab GTPase. Methods in molecular biology (Clifton, N.J.) Gomez, R. C., Vides, E. G., Pfeffer, S. R. 2021; 2293: 19-25

  Abstract

  Rab29 has been implicated in multiple membrane trafficking processes with no described effectors or regulating proteins. Its fast nucleotide exchange rate and inability to bind GDI in cytosol make it a unique and poorly understood Rab. Because the conventional, "GTP-locked" Rab mutation does not have the desired effect in Rab29, we present here the use of a fluorescence-based assay to characterize novel Rab29 mutants (I64T and V156G) that display faster nucleotide exchange rates, allowing for GEF-independent Rab29 activation.

  View details for DOI 10.1007/978-1-0716-1346-7_2

  View details for PubMedID 34453707

• Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins. eLife Saha, P. n., Shumate, J. L., Caldwell, J. G., Elghobashi-Meinhardt, N. n., Lu, A. n., Zhang, L. n., Olsson, N. E., Elias, J. E., Pfeffer, S. R. 2020; 9

  Abstract

  Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1's N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, ezetimibe, blocks NPC1L1; we show that residues that lie at the interface between NPC1L1's three extracellular domains comprise the drug's binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and ezetimibe blocks transport by binding to multiple domains simultaneously.

  View details for DOI 10.7554/eLife.57089

  View details for PubMedID 32410728

• PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins. eLife Berndsen, K., Lis, P., Yeshaw, W. M., Wawro, P. S., Nirujogi, R. S., Wightman, M., Macartney, T., Dorward, M., Knebel, A., Tonelli, F., Pfeffer, S. R., Alessi, D. R. 2019; 8

  Abstract

  Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A 'substrate-trapping' PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.

  View details for DOI 10.7554/eLife.50416

  View details for PubMedID 31663853

• Membrane association but not identity is required for LRRK2 activation and phosphorylation of Rab GTPases. The Journal of cell biology Gomez, R. C., Wawro, P., Lis, P., Alessi, D. R., Pfeffer, S. R. 2019

  Abstract

  LRRK2 kinase mutations cause familial Parkinson's disease and increased phosphorylation of a subset of Rab GTPases. Rab29 recruits LRRK2 to the trans-Golgi and activates it there, yet some of LRRK2's major Rab substrates are not on the Golgi. We sought to characterize the cell biology of LRRK2 activation. Unlike other Rab family members, we show that Rab29 binds nucleotide weakly, is poorly prenylated, and is not bound to GDI in the cytosol; nevertheless, Rab29 only activates LRRK2 when it is membrane bound and GTP bound. Mitochondrially anchored, GTP-bound Rab29 is both a LRRK2 substrate and activator, and it drives accumulation of active LRRK2 and phosphorylated Rab10 on mitochondria. Importantly, mitochondrially anchored LRRK2 is much less capable of phosphorylating plasma membrane-anchored Rab10 than soluble LRRK2. These data support a model in which LRRK2 associates with and dissociates from distinct membrane compartments to phosphorylate Rab substrates; if anchored, LRRK2 can modify misdelivered Rab substrates that then become trapped there because GDI cannot retrieve them.

  View details for DOI 10.1083/jcb.201902184

  View details for PubMedID 31624137

• NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes. The Journal of biological chemistry Pfeffer, S. R. 2019; 294 (5): 1706–9

  Abstract

  Low-density lipoprotein particles are taken up by cells and delivered to the lysosome where their cholesterol esters are cleaved off by acid lipase. The released, free cholesterol is then exported from lysosomes for cellular needs or storage. This article summarizes recent advances in our understanding of the molecular basis of cholesterol export from lysosomes. Cholesterol export requires NPC intracellular cholesterol transporter 1 (NPC1) and NPC2, genetic mutations of which can cause Niemann-Pick type C disease, a disorder characterized by massive lysosomal accumulation of cholesterol and glycosphingolipids. Analysis of the NPC1 and NPC2 structures and biochemical properties, together with new structures of the related Patched (PTCH) protein, provides new clues to the mechanisms by which NPC proteins may function.

  View details for PubMedID 30710017

• NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes JOURNAL OF BIOLOGICAL CHEMISTRY Pfeffer, S. R. 2019; 294 (5): 1706-1709

  View details for DOI 10.1074/jbc.TM118.004165

  View details for Web of Science ID 000457879200030

• Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs. eLife Lu, A., Wawro, P., Morgens, D. W., Portela, F., Bassik, M. C., Pfeffer, S. R. 2018; 7

  Abstract

  Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.

  View details for PubMedID 30556811

• Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs ELIFE Lu, A., Wawro, P., Morgens, D. W., Portela, F., Bassik, M. C., Pfeffer, S. R. 2018; 7

  View details for DOI 10.7554/eLife.41460

  View details for Web of Science ID 000454571000001

• LRRK2 and Rab GTPases BIOCHEMICAL SOCIETY TRANSACTIONS Pfeffer, S. R. 2018; 46: 1707–12

  View details for DOI 10.1042/BST20180470

  View details for Web of Science ID 000453394200028

• LRRK2 and Rab GTPases. Biochemical Society transactions Pfeffer, S. R. 2018

  Abstract

  Leucine-rich repeat kinase 2 (LRRK2) is mutated in familial Parkinson's disease, and pathogenic mutations activate the kinase activity. A tour de force screen by Mann and Alessi and co-workers identified a subset of Rab GTPases as bona fide LRRK2 substrates. Rab GTPases are master regulators of membrane trafficking and this short review will summarize what we know about the connection between LRRK2 and this family of regulatory proteins. While, in most cases, Rab GTPase phosphorylation is predicted to interfere with Rab protein function, the discovery of proteins that show preferential binding to phosphorylated Rabs suggests that more complex interactions may also contribute to mutant LRRK2-mediated pathology.

  View details for PubMedID 30467121

• A pathway for Parkinson's Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain. eLife Dhekne, H. S., Yanatori, I., Gomez, R. C., Tonelli, F., Diez, F., Schule, B., Steger, M., Alessi, D. R., Pfeffer, S. R. 2018; 7

  Abstract

  Parkinson's disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases, including Rab8A and Rab10. We show here that LRRK2 kinase interferes with primary cilia formation in cultured cells, human LRRK2 G2019S iPS cells and in the cortex of LRRK2 R1441C mice. Rab10 phosphorylation strengthens its intrinsic ability to block ciliogenesis by enhancing binding to RILPL1. Importantly, the ability of LRRK2 to interfere with ciliogenesis requires both Rab10 and RILPL1 proteins. Pathogenic LRRK2 influences the ability of cells to respond to cilia-dependent, Hedgehog signaling as monitored by Gli1 transcriptional activation. Moreover, cholinergic neurons in the striatum of LRRK2 R1441C mice show decreased ciliation, which will decrease their ability to sense Sonic hedgehog in a neuro-protective circuit that supports dopaminergic neurons. These data reveal a molecular pathway for regulating cilia function that likely contributes to Parkinson's disease-specific pathology.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

  View details for PubMedID 30398148

• A pathway for Parkinson's Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain ELIFE Dhekne, H. S., Yanatori, I., Gomez, R. C., Tonelli, F., Diez, F., Schule, B., Steger, M., Alessi, D. R., Pfeffer, S. R. 2018; 7

  View details for DOI 10.7554/eLife.40202

  View details for Web of Science ID 000458161900001

• Rab29 activation of the Parkinson's disease-associated LRRK2 kinase EMBO JOURNAL Purlyte, E., Dhekne, H. S., Sarhan, A. R., Gomez, R., Lis, P., Wightman, M., Martinez, T. N., Tonelli, F., Pfeffer, S. R., Alessi, D. R. 2018; 37 (1): 1–18

  Abstract

  Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

  View details for PubMedID 29212815

  View details for PubMedCentralID PMC5753036

• Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis ELIFE Steger, M., Diez, F., Dhekne, H. S., Lis, P., Nirujogi, R. S., Karayel, O., Tonelli, F., Martinez, T. N., Lorentzen, E., Pfeffer, S. R., Alessi, D. R., Mann, M. 2017; 6

  Abstract

  We previously reported that Parkinson's disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

  View details for PubMedID 29125462

• NPC1-mediated cholesterol export from lysosomes Pfeffer, S. R. FEDERATION AMER SOC EXP BIOL. 2017

  View details for Web of Science ID 000405461402528

• Rab GTPases: master regulators that establish the secretory and endocytic pathways MOLECULAR BIOLOGY OF THE CELL Pfeffer, S. R. 2017; 28 (6): 712-715

  Abstract

  Several of the most important discoveries in the field of membrane traffic have come from studies of Rab GTPases by Marino Zerial and Peter Novick and their colleagues. Zerial was the first to discover that Rab GTPases represent identity markers for different membrane-bound compartments, and each Rab organizes a collection of specific effectors into function-specifying membrane microdomains to carry out receptor trafficking. Novick discovered that the order (and thus polarity) of Rab GTPases along the secretory and endocytic pathways are established by their specific, cognate guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which partner with one Rab to regulate the subsequent- and prior-acting Rabs. Such so-called Rab cascades have evolved to establish domains that contain unique Rab proteins and their cognate effectors, which drive all steps of membrane trafficking. These findings deserve much broader recognition by the biomedical research community and are highlighted here, along with open questions that require serious attention for full understanding of the molecular basis of Rab GTPase-regulated membrane trafficking in eukaryotic cells.

  View details for DOI 10.1091/mbc.E16-10-0737

  View details for Web of Science ID 000396240700002

  View details for PubMedID 28292916

• Quantitative Measurement of Cholesterol in Cell Populations Using Flow Cytometry and Fluorescent Perfringolysin O. Methods in molecular biology (Clifton, N.J.) Li, J., Lee, P. L., Pfeffer, S. R. 2017; 1583: 85-95

  Abstract

  Methods to quantify intracellular cholesterol are valuable for the study of its trafficking and storage in normal cells and in lysosomal storage disorders. Traditionally, cholesterol has been tracked using the small molecule, filipin. Filipin can be difficult to visualize and visualization can be cytotoxic as it requires UV illumination. Here we describe a method to measure cholesterol using a fluorescently labeled, mutant form of Perfringolysin O, a soluble protein toxin that binds cholesterol specifically. This approach has been used to measure the impact of NPC1 deficiency on lysosomal cholesterol levels and monitor the rescue of cholesterol export under conditions that reduce the thickness of the lysosomal glycocalyx.

  View details for DOI 10.1007/978-1-4939-6875-6_8

  View details for PubMedID 28205169

• Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export. eLife Li, J., Pfeffer, S. R. 2016; 5

  Abstract

  LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon's disease, an X-linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone-mediated autophagy, and its expression improves tissue function in models of aging. We show here that human LAMP1 and LAMP2 bind cholesterol in a manner that buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly.

  View details for DOI 10.7554/eLife.21635

  View details for PubMedID 27664420

• Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2. Proceedings of the National Academy of Sciences of the United States of America Li, X., Saha, P., Li, J., Blobel, G., Pfeffer, S. R. 2016; 113 (36): 10079-10084

  Abstract

  Export of LDL-derived cholesterol from lysosomes requires the cooperation of the integral membrane protein Niemann-Pick C1 (NPC1) and a soluble protein, Niemann-Pick C2 (NPC2). Mutations in the genes encoding these proteins lead to Niemann-Pick disease type C (NPC). NPC2 binds to NPC1's second (middle), lumenally oriented domain (MLD) and transfers cholesterol to NPC1's N-terminal domain (NTD). Here, we report the 2.4-Å resolution crystal structure of a complex of human NPC1-MLD and NPC2 bearing bound cholesterol-3-O-sulfate. NPC1-MLD uses two protruding loops to bind NPC2, analogous to its interaction with the primed Ebola virus glycoprotein. Docking of the NPC1-NPC2 complex onto the full-length NPC1 structure reveals a direct cholesterol transfer tunnel between NPC2 and NTD cholesterol binding pockets, supporting the "hydrophobic hand-off" cholesterol transfer model.

  View details for DOI 10.1073/pnas.1611956113

  View details for PubMedID 27551080

  View details for PubMedCentralID PMC5018801

• Clues to NPC1-mediated cholesterol export from lysosomes. Proceedings of the National Academy of Sciences of the United States of America Pfeffer, S. R. 2016; 113 (29): 7941-7943

  View details for DOI 10.1073/pnas.1608530113

  View details for PubMedID 27410046

  View details for PubMedCentralID PMC4961152

• Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis MOLECULAR BIOLOGY OF THE CELL Johnson, T. A., Pfeffer, S. R. 2016; 27 (11): 1845-1852

  Abstract

  Human NPC1L1 protein mediates cholesterol absorption in the intestine and liver and is the target of the drug ezetimibe, which is used to treat hypercholesterolemia. Previous studies concluded that NPC1L1-GFP protein trafficking is regulated by cholesterol binding and that ezetimibe blocks NPC1L1-GFP function by inhibiting its endocytosis. We used cell surface biotinylation to monitor NPC1L1-GFP endocytosis and show that ezetimibe does not alter the rate of NPC1L1-GFP endocytosis in cultured rat hepatocytes grown under normal growth conditions. As expected, NPC1L1-GFP endocytosis depends in part on C-terminal, cytoplasmically oriented sequences, but endocytosis does not require cholesterol binding to NPC1L1's N-terminal domain. In addition, two small- molecule inhibitors of general (and NPC1L1-GFP) endocytosis failed to inhibit the ezetimibe-sensitive uptake of [(3)H]cholesterol from taurocholate micelles. These experiments demonstrate that cholesterol uptake by NPC1L1 does not require endocytosis; moreover, ezetimibe interferes with NPC1L1's cholesterol adsorption activity without blocking NPC1L1 internalization in RH7777 cells.

  View details for DOI 10.1091/mbc.E16-03-0154

  View details for Web of Science ID 000376777600014

  View details for PubMedID 27075173

  View details for PubMedCentralID PMC4884074

• Lipoprotein secretion: It takes two to TANGO. The Journal of cell biology Pfeffer, S. R. 2016

  Abstract

  An unsolved mystery in cell biology is how unusually large secretory cargoes are exported from the endoplasmic reticulum. In this issue, Santos et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201603072) report the function of a Mia2/cTAGE5 transcript fusion, named TALI, in the endoplasmic reticulum export of chylomicrons and very low-density lipoproteins, but not collagen XII.

  View details for PubMedID 27138249

• LAMP proteins bind cholesterol and contribute to NPC1-mediated cholesterol export from lysosomes. Li, J., Pfeffer, S. R. AMER SOC CELL BIOLOGY. 2016

  View details for Web of Science ID 000394259500084

• Transport Vesicle Tethering at the Trans Golgi Network: Coiled Coil Proteins in Action. Frontiers in cell and developmental biology Cheung, P. P., Pfeffer, S. R. 2016; 4: 18-?

  Abstract

  The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed.

  View details for DOI 10.3389/fcell.2016.00018

  View details for PubMedID 27014693

  View details for PubMedCentralID PMC4791371

• Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells. Proceedings of the National Academy of Sciences of the United States of America Li, J., Deffieu, M. S., Lee, P. L., Saha, P., Pfeffer, S. R. 2015; 112 (48): 14876-14881

  Abstract

  Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [(14)C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.

  View details for DOI 10.1073/pnas.1520490112

  View details for PubMedID 26578804

  View details for PubMedCentralID PMC4672801

• The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization ELIFE Lee, P. L., Ohlson, M. B., Pfeffer, S. R. 2015; 4

  Abstract

  Most kinesins transport cargoes bound to their C-termini and use N-terminal motor domains to move along microtubules. We report here a novel function for KIF1C: it transports Rab6A-vesicles and can influence Golgi complex organization. These activities correlate with KIF1C's capacity to bind the Golgi protein Rab6A directly, both via its motor domain and C-terminus. Rab6A binding to the motor domain inhibits microtubule interaction in vitro and in cells, decreasing the amount of motile KIF1C. KIF1C depletion slows protein delivery to the cell surface, interferes with vesicle motility, and triggers Golgi fragmentation. KIF1C can protect Golgi membranes from fragmentation in cells lacking an intact microtubule network. Rescue of fragmentation requires sequences that enable KIF1C to bind Rab6A at both ends, but not KIF1C motor function. Rab6A binding to KIF1C's motor domain represents an entirely new mode of regulation for a kinesin motor, and likely has important consequences for KIF1C's cellular functions.

  View details for DOI 10.7554/eLife.06029

  View details for Web of Science ID 000373785900001

  View details for PubMedCentralID PMC4405695

• Measuring Rab GTPase-Activating Protein (GAP) Activity in Live Cells and Extracts. Methods in molecular biology (Clifton, N.J.) Nottingham, R. M., Pfeffer, S. R. 2015; 1298: 61-71

  Abstract

  Mammalian cells encode a diverse set of Rab GTPases and their corresponding regulators. In vitro biochemical screening has proven invaluable in assigning particular Rabs as substrates for their cognate GTPase-activating proteins. However, in vitro activity does not always reflect substrate specificity in cells. This method describes a functional test of GAP activity in cells or extracts that takes into account the presence of other factors or conditions that might change observed in vitro specificity.

  View details for DOI 10.1007/978-1-4939-2569-8_5

  View details for PubMedID 25800832

• Protein flexibility is required for vesicle tethering at the Golgi. eLife Cheung, P. P., Limouse, C., Mabuchi, H., Pfeffer, S. R. 2015; 4

  Abstract

  The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185's ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185's N-and C-termini are within.

  View details for DOI 10.7554/eLife.12790

  View details for PubMedID 26653856

  View details for PubMedCentralID PMC4721967

• Rab6 regulation of the kinesin family KIF1C motor domain contributes to Golgi tethering. eLife Lee, P. L., Ohlson, M. B., Pfeffer, S. R. 2015; 4

  Abstract

  Most kinesins transport cargoes bound to their C-termini and use N-terminal motor domains to move along microtubules. We report here a novel function for KIF1C: it transports Rab6A-vesicles and can influence Golgi complex organization. These activities correlate with KIF1C's capacity to bind the Golgi protein Rab6A directly, both via its motor domain and C-terminus. Rab6A binding to the motor domain inhibits microtubule interaction in vitro and in cells, decreasing the amount of motile KIF1C. KIF1C depletion slows protein delivery to the cell surface, interferes with vesicle motility, and triggers Golgi fragmentation. KIF1C can protect Golgi membranes from fragmentation in cells lacking an intact microtubule network. Rescue of fragmentation requires sequences that enable KIF1C to bind Rab6A at both ends, but not KIF1C motor function. Rab6A binding to KIF1C's motor domain represents an entirely new mode of regulation for a kinesin motor, and likely has important consequences for KIF1C's cellular functions.

  View details for DOI 10.7554/eLife.06029

  View details for PubMedID 25821985

  View details for PubMedCentralID PMC4405695

• Molecular and Cellular Characterization of GCC185: A Tethering Protein of the Trans-Golgi Network. Methods in molecular biology (Clifton, N.J.) Cheung, P. P., Pfeffer, S. R. 2015; 1270: 179-190

  Abstract

  Transport vesicle tethers are proteins that link partner membranes together to permit subsequent SNARE protein pairing and fusion. Despite the identification of a relatively large number of tethering proteins, little is known about the precise mechanisms by which they act. Biochemical isolation of tethers permits direct analysis of their physical characteristics and molecular interactions. Here, we describe the expression and purification of GCC185, a trans-Golgi-localized, 190-kDa coiled-coil tethering protein. In addition, we present a gene rescue approach to analyze the function of this tether after its depletion from cells using siRNA.

  View details for DOI 10.1007/978-1-4939-2309-0_14

  View details for PubMedID 25702118

• Conformational flexibility of GCC185 is required for vesicle tethering at the trans Golgi. Cheung, P., Limouse, C., Mabuchi, H., Pfeffer, S. R. AMER SOC CELL BIOLOGY. 2014

  View details for Web of Science ID 000352094105028

• A CULLINary ride across the secretory pathway: more than just secretion TRENDS IN CELL BIOLOGY Lu, A., Pfeffer, S. R. 2014; 24 (7): 389-399

  Abstract

  Mulitmeric cullin-RING ubiquitin ligases (CRLs) represent the largest class of ubiquitin ligases in eukaryotes. However, most CRL ubiquitylation pathways remain uncharacterized. CRLs control a myriad of functions by catalyzing mono- or poly-ubiquitylation of target proteins. Recently, novel CRLs have been identified along the secretory pathway where they modify substrates involved in diverse cellular processes such as vesicle coat assembly and cell cycle progression. This review discusses our current understanding of CRL ubiquitylation within the secretory pathway, with special emphasis on the emerging role of the Golgi as a ubiquitylation platform. CRLs are also implicated in endosome function, where their specific roles are less well understood.

  View details for DOI 10.1016/j.tcb.2014.02.001

  View details for Web of Science ID 000338410900002

  View details for PubMedID 24630736

  View details for PubMedCentralID PMC4074570

• Mutant enzymes challenge all assumptions. eLife Nottingham, R. M., Pfeffer, S. R. 2014; 3

  Abstract

  Enzymes called Rab GTPases that carry so-called "activating" mutations may never become activated at all.

  View details for DOI 10.7554/eLife.02171

  View details for PubMedID 24520166

  View details for PubMedCentralID PMC3919269

• A Prize for Membrane Magic CELL Pfeffer, S. R. 2013; 155 (6): 1203-1206

  Abstract

  The 2013 Nobel Prize in Physiology or Medicine has been awarded to James Rothman, Randy Schekman, and Thomas Südhof "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells". I present a personal view of the membrane trafficking field, highlighting the contributions of these three Nobel laureates in a historical context.

  View details for DOI 10.1016/j.cell.2013.11.014

  View details for Web of Science ID 000328271100002

  View details for PubMedID 24315088

• Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression JOURNAL OF CELL BIOLOGY Lu, A., Pfeffer, S. R. 2013; 203 (2): 233-250

  Abstract

  Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated cyclin E levels. On the Golgi, RhoBTB3 bound cyclin E as part of a Cullin3 (CUL3)-dependent RING-E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.

  View details for DOI 10.1083/jcb.201305158

  View details for Web of Science ID 000326281200009

  View details for PubMedID 24145166

  View details for PubMedCentralID PMC3812982

• Rab GTPase regulation of membrane identity CURRENT OPINION IN CELL BIOLOGY Pfeffer, S. R. 2013; 25 (4): 414-419

  Abstract

  A fundamental question in cell biology is how cells determine membrane compartment identity and the directionality with which cargoes pass through the secretory and endocytic pathways. The discovery of so-called 'Rab cascades' provides a satisfying molecular mechanism that helps to resolve this paradox. One Rab GTPase has the ability to template the localization of the subsequent acting Rab GTPase along a given transport pathway. Thus, in addition to determining compartment identity and functionality, Rab GTPases are likely able to order the events of membrane trafficking. This review will highlight recent advances in our understanding of Rabs and Rab cascades.

  View details for DOI 10.1016/j.ceb.2013.04.002

  View details for Web of Science ID 000323084700003

  View details for PubMedID 23639309

• A nexus for receptor recycling. Nature cell biology Pfeffer, S. R. 2013; 15 (5): 446-448

  Abstract

  Sorting nexin proteins (SNXs) and the cargo-selective retromer complex play key roles in receptor recycling from endosomes to the cell surface. A global proteomics analysis reveals a collection of cell surface proteins that rely on SNX27 and the retromer complex for their cell surface localization at steady state.

  View details for DOI 10.1038/ncb2751

  View details for PubMedID 23636423

• Hopping rim to rim through the Golgi. eLife Pfeffer, S. R. 2013; 2

  Abstract

  A novel approach based on tracking the fate of proteins that become 'stapled' to the walls of the Golgi yields insights into the long-sought mechanism of transport through this organelle.

  View details for DOI 10.7554/eLife.00903

  View details for PubMedID 23795298

  View details for PubMedCentralID PMC3679515

• Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase JOURNAL OF BIOLOGICAL CHEMISTRY Pusapati, G. V., Luchetti, G., Pfeffer, S. R. 2012; 287 (50): 42129-42137

  Abstract

  Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.

  View details for DOI 10.1074/jbc.M112.414565

  View details for Web of Science ID 000312103000051

  View details for PubMedID 23091056

  View details for PubMedCentralID PMC3516758

• Rab GTPase localization and Rab cascades in Golgi transport BIOCHEMICAL SOCIETY TRANSACTIONS Pfeffer, S. R. 2012; 40: 1373-1377

  Abstract

  Rab GTPases are master regulators of membrane traffic. By binding to distinct sets of effector proteins, Rabs catalyse the formation of function-specifying membrane microdomains. They are delivered to membranes by a protein named GDI (guanine-nucleotide-dissociation inhibitor) and are stabilized there after nucleotide exchange by effector binding. In the present mini-review, I discuss what we know about how Rab GTPases are delivered to the correct membrane-bound compartments and how Rab GTPase cascades order Rabs within the secretory and endocytic pathways. Finally, I describe how Rab cascades may establish the distinct compartments of the Golgi complex to permit ordered processing, sorting and secretion of secretory cargoes.

  View details for DOI 10.1042/BST20120168

  View details for Web of Science ID 000312096800036

  View details for PubMedID 23176483

• The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module HUMAN MOLECULAR GENETICS van Rahden, V. A., Brand, K., Najm, J., Heeren, J., Pfeffer, S. R., Braulke, T., Kutsche, K. 2012; 21 (23): 5019-5038

  Abstract

  Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.

  View details for DOI 10.1093/hmg/dds343

  View details for Web of Science ID 000310967900001

  View details for PubMedID 22907655

  View details for PubMedCentralID PMC3490508

• Cargo carriers from the Golgi to the cell surface EMBO JOURNAL Pfeffer, S. R. 2012; 31 (20): 3954-3955

  Abstract

  In this issue, Malhotra and colleagues use biochemical approaches to identify a new class of secretory cargo carriers (CARTS) that do not contain the larger cargoes, collagen or Vesicular stomatitis virus (VSV)-G glycoprotein. CARTS appear to be basolateral membrane-directed carriers that use myosin for their motility but not for their formation.

  View details for DOI 10.1038/emboj.2012.249

  View details for Web of Science ID 000310055400002

  View details for PubMedID 22940689

  View details for PubMedCentralID PMC3474924

• TBC1D16 is a Rab4A GTPase activating protein that regulates receptor recycling and EGF receptor signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Goueli, B. S., Powell, M. B., Finger, E. C., Pfeffer, S. R. 2012; 109 (39): 15787-15792

  Abstract

  Rab4A is a master regulator of receptor recycling from endocytic compartments to the plasma membrane. The protein TBC1D16 is up-regulated in melanoma, and TBC1D16-overexpressing melanoma cells are dependent on TBC1D16. We show here that TBC1D16 enhances the intrinsic rate of GTP hydrolysis by Rab4A. TBC1D16 is both cytosolic and membrane associated; the membrane-associated pool colocalizes with transferrin and EGF receptors (EGFRs) and early endosome antigen 1, but not with LAMP1 protein. Expression of two TBC1D16 isoforms, but not the inactive R494A mutant, reduces transferrin receptor recycling but has no effect on transferrin receptor internalization. Expression of TBC1D16 alters GFP-Rab4A membrane localization. In HeLa cells, overexpression of TBC1D16 enhances EGF-stimulated EGFR degradation, concomitant with decreased EGFR levels and signaling. Thus, TBC1D16 is a GTPase activating protein for Rab4A that regulates transferrin receptor recycling and EGFR trafficking and signaling.

  View details for DOI 10.1073/pnas.1204540109

  View details for Web of Science ID 000309604500056

  View details for PubMedID 23019362

  View details for PubMedCentralID PMC3465424

• RUTBC2 Protein, a Rab9A Effector and GTPase-activating Protein for Rab36 JOURNAL OF BIOLOGICAL CHEMISTRY Nottingham, R. M., Pusapati, G. V., Ganley, I. G., Barr, F. A., Lambright, D. G., Pfeffer, S. R. 2012; 287 (27): 22740-22748

  Abstract

  Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.

  View details for DOI 10.1074/jbc.M112.362558

  View details for Web of Science ID 000306495000031

  View details for PubMedID 22637480

  View details for PubMedCentralID PMC3391118

• Ebola virus entry requires the host-programmed recognition of an intracellular receptor EMBO JOURNAL Miller, E. H., Obernosterer, G., Raaben, M., Herbert, A. S., Deffieu, M. S., Krishnan, A., Ndungo, E., Sandesara, R. G., Carette, J. E., Kuehne, A. I., Ruthel, G., Pfeffer, S. R., Dye, J. M., Whelan, S. P., Brummelkamp, T. R., Chandran, K. 2012; 31 (8): 1947-1960

  Abstract

  Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.

  View details for DOI 10.1038/emboj.2012.53

  View details for Web of Science ID 000303108600010

  View details for PubMedID 22395071

  View details for PubMedCentralID PMC3343336

• Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deffieu, M. S., Pfeffer, S. R. 2011; 108 (47): 18932-18936

  Abstract

  Niemann-Pick type C1 (NPC1) protein is needed for cellular utilization of low-density lipoprotein-derived cholesterol that has been delivered to lysosomes. The protein has 13 transmembrane domains, three large lumenal domains, and a cytoplasmic tail. NPC1's lumenally oriented, N-terminal domain binds cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of the process by which cholesterol is exported from lysosomes into the cytosol. Using surface plasmon resonance and affinity chromatography, we show here that the second lumenal domain of NPC1 binds directly to NPC2 protein. For these experiments, a soluble NPC1 lumenal domain 2 was engineered by replacing adjacent transmembrane domains with antiparallel coiled-coil sequences. Interaction of NPC2 with NPC1 lumenal domain 2 is only detected at acidic pH, conditions that are optimal for cholesterol binding to NPC2 and transfer to NPC1; the pH is also appropriate for the acidic environment where binding would take place. Binding to NPC1 domain 2 requires the presence of cholesterol on NPC2 protein, a finding that supports directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain. Finally, human disease-causing mutations in NPC1 domain 2 decrease NPC2 binding, suggesting that NPC2 binding is necessary for NPC1 function in humans. These data support a model in which NPC1 domain 2 holds NPC2 in position to facilitate directional cholesterol transfer from NPC2 onto NPC1 protein for export from lysosomes.

  View details for DOI 10.1073/pnas.1110439108

  View details for Web of Science ID 000297249800022

  View details for PubMedID 22065762

  View details for PubMedCentralID PMC3223457

• RUTBC1 Protein, a Rab9A Effector That Activates GTP Hydrolysis by Rab32 and Rab33B Proteins JOURNAL OF BIOLOGICAL CHEMISTRY Nottingham, R. M., Ganley, I. G., Barr, F. A., Lambright, D. G., Pfeffer, S. R. 2011; 286 (38): 33213-33222

  Abstract

  Rab GTPases regulate all steps of membrane trafficking. Their interconversion between active, GTP-bound states and inactive, GDP-bound states is regulated by guanine nucleotide exchange factors and GTPase-activating proteins. The substrates for most Rab GTPase-activating proteins (GAPs) are unknown. Rab9A and its effectors regulate transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. We show here that RUTBC1 is a Tre2/Bub2/Cdc16 domain-containing protein that binds to Rab9A-GTP both in vitro and in cultured cells, but is not a GTPase-activating protein for Rab9A. Biochemical screening of RUTBC1 Rab protein substrates revealed highest in vitro GTP hydrolysis-activating activity with Rab32 and Rab33B. Catalysis required Arg-803 of RUTBC1, and RUTBC1 could activate a catalytically inhibited Rab33B mutant (Q92A), in support of a dual finger mechanism for RUTBC1 action. Rab9A binding did not influence GAP activity of bead-bound RUTBC1 protein. In cells and cell extracts, RUTBC1 influenced the ability of Rab32 to bind its effector protein, Varp, consistent with a physiological role for RUTBC1 in regulating Rab32. In contrast, binding of Rab33B to its effector protein, Atg16L1, was not influenced by RUTBC1 in cells or extracts. The identification of a protein that binds Rab9A and inactivates Rab32 supports a model in which Rab9A and Rab32 act in adjacent pathways at the boundary between late endosomes and the biogenesis of lysosome-related organelles.

  View details for DOI 10.1074/jbc.M111.261115

  View details for Web of Science ID 000294968800040

  View details for PubMedID 21808068

  View details for PubMedCentralID PMC3190867

• GCC185 plays independent roles in Golgi structure maintenance and AP-1-mediated vesicle tethering JOURNAL OF CELL BIOLOGY Brown, F. C., Schindelhaim, C. H., Pfeffer, S. R. 2011; 194 (5): 779-787

  Abstract

  GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)-containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.

  View details for DOI 10.1083/jcb.201104019

  View details for Web of Science ID 000294602500013

  View details for PubMedID 21875948

  View details for PubMedCentralID PMC3171126

• Entry at the trans-Face of the Golgi COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Pfeffer, S. R. 2011; 3 (3)

  Abstract

  The trans-Golgi network (TGN) receives a select set of proteins from the endocytic pathway-about 5% of total plasma membrane glycoproteins (Duncan and Kornfeld 1988). Proteins that are delivered include mannose 6-phosphate receptors (MPRs), TGN46, sortilin, and various toxins that hitchhike a ride backward through the secretory pathway to intoxicate cells after they exit into the cytoplasm from the endoplasmic reticulum (ER). This article will review work on the molecular players that drive protein transport from the endocytic pathway to the TGN. Distinct requirements have revealed multiple routes for retrograde transport; in addition, the existence of multiple, potential coat proteins and/or cargo adaptors imply that multiple vesicular transfers are likely involved. Several comprehensive reviews have appeared recently and should be sought for additional details (Bonifacino and Rojas 2006; Johannes and Popoff 2008).

  View details for DOI 10.1101/cshperspect.a005272

  View details for Web of Science ID 000287846200004

  View details for PubMedID 21421921

  View details for PubMedCentralID PMC3039930

• How the Golgi works: A cisternal progenitor model PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pfeffer, S. R. 2010; 107 (46): 19614-19618

  Abstract

  The Golgi complex is a central processing compartment in the secretory pathway of eukaryotic cells. This essential compartment processes more than 30% of the proteins encoded by the human genome, yet we still do not fully understand how the Golgi is assembled and how proteins pass through it. Recent advances in our understanding of the molecular basis for protein transport through the Golgi and within the endocytic pathway provide clues to how this complex organelle may function and how proteins may be transported through it. Described here is a possible model for transport of cargo through a tightly stacked Golgi that involves continual fusion and fission of stable, "like" subcompartments and provides a mechanism to grow the Golgi complex from a stable progenitor, in an ordered manner.

  View details for DOI 10.1073/pnas.1011016107

  View details for Web of Science ID 000284261800007

  View details for PubMedID 21045128

  View details for PubMedCentralID PMC2993360

• An update on transport vesicle tethering MOLECULAR MEMBRANE BIOLOGY Brown, F. C., Pfeffer, S. R. 2010; 27 (8): 457-461

  Abstract

  Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.

  View details for DOI 10.3109/09687688.2010.501765

  View details for Web of Science ID 000285246100007

  View details for PubMedID 21067454

• Membrane traffic Editorial overview CURRENT OPINION IN CELL BIOLOGY Pfeffer, S. R., Novick, P. J. 2010; 22 (4): 419-421

  View details for DOI 10.1016/j.ceb.2010.06.001

  View details for Web of Science ID 000280945500001

  View details for PubMedID 20566276

• F1000 How Does Your Golgi Go? SCIENTIST Pfeffer, S. R. 2010; 24 (5): 65-66

  View details for Web of Science ID 000277037900021

• Unconventional secretion by autophagosome exocytosis JOURNAL OF CELL BIOLOGY Pfeffer, S. R. 2010; 188 (4): 451-452

  Abstract

  In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)-binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway.

  View details for DOI 10.1083/jcb.201001121

  View details for Web of Science ID 000274723800003

  View details for PubMedID 20156968

  View details for PubMedCentralID PMC2828920

• Recent advances in understanding Golgi biogenesis. F1000 biology reports Pfeffer, S. R. 2010; 2: 32-?

  Abstract

  The Golgi complex is a central processing station for proteins traversing the secretory pathway, yet we are still learning how this compartment is constructed and how cargo moves through it. Recent experiments suggest a key role for Ras-like Rab GTPases and provide important new ideas for how the Golgi may function.

  View details for DOI 10.3410/B2-32

  View details for PubMedID 20625450

  View details for PubMedCentralID PMC2897732

• Two Rabs for exosome release NATURE CELL BIOLOGY Pfeffer, S. R. 2010; 12 (1): 3-4

  Abstract

  Exosomes are endosome-derived membrane vesicles that are key for intercellular communication in the immune system and elsewhere. Rab27A and Rab27B GTPases and two of their cognate effector proteins seem to be needed to drive the physiologically important exosome-release process in certain cell types.

  View details for DOI 10.1038/ncb0110-3

  View details for Web of Science ID 000272973800004

  View details for PubMedID 20027197

• Multiple routes of protein transport from endosomes to the trans Golgi etwork FEBS LETTERS Pfeffer, S. R. 2009; 583 (23): 3811-3816

  Abstract

  Proteins use multiple routes for transport from endosomes to the Golgi complex. Shiga and cholera toxins and TGN38/46 are routed from early and recycling endosomes, while mannose 6-phosphate receptors are routed from late endosomes. The identification of distinct molecular requirements for each of these pathways makes it clear that mammalian cells have evolved more complex targeting mechanisms and routes than previously anticipated.

  View details for DOI 10.1016/j.febslet.2009.10.075

  View details for Web of Science ID 000272421200012

  View details for PubMedID 19879268

  View details for PubMedCentralID PMC2787657

• Defining the boundaries: Rab GEFs and GAPs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nottingham, R. M., Pfeffer, S. R. 2009; 106 (34): 14185-14186

  View details for DOI 10.1073/pnas.0907725106

  View details for Web of Science ID 000269295100005

  View details for PubMedID 19706500

  View details for PubMedCentralID PMC2732820

• RhoBTB3: A Rho GTPase-Family ATPase Required for Endosome to Golgi Transport CELL Espinosa, E. J., Calero, M., Sridevi, K., Pfeffer, S. R. 2009; 137 (5): 938-948

  Abstract

  Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3's N-terminal, Rho-related domain and C-terminal sequences that are important for Rab9 interaction. Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the autoinhibited RhoBTB3 protein to permit maximal ATP hydrolysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.

  View details for DOI 10.1016/j.cell.2009.03.043

  View details for Web of Science ID 000266454800023

  View details for PubMedID 19490898

  View details for PubMedCentralID PMC2801561

• Roles for Rab6, Arl1 and a novel Rho protein in GCC185-mediated vesicle tethering at the trans Golgi network Pfeffer, S. R. FEDERATION AMER SOC EXP BIOL. 2009

  View details for Web of Science ID 000208621500050

• Rab9 regulation of the Rab GTPase activating protein, RUTBC1 Nottingham, R. M., Ganley, I. G., Barr, F. A., Lambright, D. G., Pfeffer, S. R. FEDERATION AMER SOC EXP BIOL. 2009

  View details for Web of Science ID 000208621504633

• Multiple Rab GTPase Binding Sites in GCC185 Suggest a Model for Vesicle Tethering at the Trans-Golgi MOLECULAR BIOLOGY OF THE CELL Hayes, G. L., Brown, F. C., Haas, A. K., Nottingham, R. M., Barr, F. A., Pfeffer, S. R. 2009; 20 (1): 209-217

  Abstract

  GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.

  View details for DOI 10.1091/mbc.E08-07-0740

  View details for Web of Science ID 000262134800020

  View details for PubMedID 18946081

  View details for PubMedCentralID PMC2613123

• WHAMMing into the Golgi DEVELOPMENTAL CELL Hayes, G. L., Pfeffer, S. R. 2008; 15 (2): 171-172

  Abstract

  A new paper from Campellone et al. in a recent issue of Cell identifies WHAMM, a multifunctional protein that stimulates Arp2/3-mediated actin polymerization, binds and organizes microtubules, and influences the structure and efficiency of the Golgi complex. WHAMM's membrane localization at the entry face of the Golgi complex is novel for an actin nucleation-promoting factor, and highlights the importance of the cytoskeleton in organizing the secretory pathway.

  View details for DOI 10.1016/j.devcel.2008.07.011

  View details for Web of Science ID 000258545900001

  View details for PubMedID 18694552

• Team effort by TRAPP forces a nucleotide fumble CELL Nottingham, R. M., Pfeffer, S. R. 2008; 133 (7): 1141-1143

  Abstract

  TRAPPI is a multisubunit protein complex on the Golgi that activates the small GTPase Ypt1p to facilitate the receipt of transport vesicles inbound from the endoplasmic reticulum. Cai et al. (2008) now present structural and biochemical analyses of yeast TRAPPI in a complex with Ypt1p revealing a unique mechanism by which TRAPPI catalyzes guanine nucleotide exchange.

  View details for DOI 10.1016/j.cell.2008.06.012

  View details for Web of Science ID 000257144600011

  View details for PubMedID 18585348

• Rab and arl GTPase family members cooperate in the localization of the golgin GCC185 CELL Burguete, A. S., Fenn, T. D., Brunger, A. T., Pfeffer, S. R. 2008; 132 (2): 286-298

  Abstract

  GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.

  View details for DOI 10.1016/j.cell.2007.11.048

  View details for Web of Science ID 000253427700014

  View details for PubMedID 18243103

  View details for PubMedCentralID PMC2344137

• A syntaxin 10-SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells JOURNAL OF CELL BIOLOGY Ganley, I. G., Espinosa, E., Pfeffer, S. R. 2008; 180 (1): 159-172

  Abstract

  Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE-tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.

  View details for Web of Science ID 000252746800016

  View details for PubMedID 18195106

• A tribute to Arthur Kornberg 1918-2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY Fuller, R. S., Bambara, R. A., Baker, T., Funnell, B., Wahle, E., O'Donnell, M., Kaiser, D., Skarstad, K., Konforti, B., Maki, S., Katayama, T., Sekimizu, K., Weiner, J. H., Davis, R. W., Rowen, L., Goodman, M. F., Spudich, J., Pfeffer, S., Richardson, C. C., Polaczek, P., Calendar, R., Kolodner, R., Griffith, J., Stillman, B., Modrich, P., Brenner, C., Yanofsky, C. 2008; 15 (1): 2–17

  View details for Web of Science ID 000252118800002

  View details for PubMedID 18176549

• TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication JOURNAL OF BIOLOGICAL CHEMISTRY Sklan, E. H., Serrano, R. L., Einav, S., Pfeffer, S. R., Lambright, D. G., Glenn, J. S. 2007; 282 (50): 36354-36361

  Abstract

  Like other viruses, productive hepatitis C virus (HCV) infection depends on certain critical host factors. We have recently shown that an interaction between HCV nonstructural protein NS5A and a host protein, TBC1D20, is necessary for efficient HCV replication. TBC1D20 contains a TBC (Tre-2, Bub2, and Cdc16) domain present in most known Rab GTPase-activating proteins (GAPs). The latter are master regulators of vesicular membrane transport, as they control the activity of membrane-associated Rab proteins. To better understand the role of the NS5A-TBC1D20 interaction in the HCV life cycle, we used a biochemical screen to identify the TBC1D20 Rab substrate. TBC1D20 was found to be the first known GAP for Rab1, which is implicated in the regulation of anterograde traffic between the endoplasmic reticulum and the Golgi complex. Mutation of amino acids implicated in Rab GTPase activation by other TBC domain-containing GAPs abrogated the ability of TBC1D20 to activate Rab1 GTPase. Overexpression of TBC1D20 blocked the transport of exogenous vesicular stomatitis virus G protein from the endoplasmic reticulum, validating the involvement of TBC1D20 in this pathway. Rab1 depletion significantly decreased HCV RNA levels, suggesting a role for Rab1 in HCV replication. These results highlight a novel mechanism by which viruses can hijack host cell machinery and suggest an attractive model whereby the NS5A-TBC1D20 interaction may promote viral membrane-associated RNA replication.

  View details for DOI 10.1074/jbc.M705221200

  View details for PubMedID 17901050

• Microbiology - Pathogen drop-kick NATURE Pfeffer, S. 2007; 450 (7168): 361-362

  View details for DOI 10.1038/450361a

  View details for Web of Science ID 000250918600035

  View details for PubMedID 18004371

• Unsolved mysteries in membrane traffic ANNUAL REVIEW OF BIOCHEMISTRY Pfeffer, S. R. 2007; 76: 629-645

  Abstract

  Remarkable strides have been made over the past 20 years in elucidating the molecular basis of membrane trafficking. Indeed, a combination of biochemical and genetic approaches have determined the identity and function of many of the core constituents needed for protein secretion and endocytosis. But much remains to be learned. This review highlights underlying themes in membrane traffic to help us refocus and solve many remaining and newly emerging issues that are fundamental to mammalian cell biology and human physiology.

  View details for DOI 10.1146/annurev.biochem.76.061705.130002

  View details for Web of Science ID 000249336800026

  View details for PubMedID 17263661

• Clues to Neuro-Degeneration in Niemann-Pick Type C Disease from Global Gene Expression Profiling PLOS ONE Reddy, J. V., Ganley, I. G., Pfeffer, S. R. 2006; 1 (1)

  Abstract

  Niemann-Pick Type C (NPC) disease is a neurodegenerative disease that is characterized by the accumulation of cholesterol and glycosphingolipids in the late endocytic pathway. The majority of NPC cases are due to mutations in the NPC1 gene. The precise function of this gene is not yet known.Using cDNA microarrays, we analyzed the genome-wide expression patterns of human fibroblasts homozygous for the I1061T NPC1 mutation that is characterized by a severe defect in the intracellular processing of low density lipoprotein-derived cholesterol. A distinct gene expression profile was identified in NPC fibroblasts from different individuals when compared with fibroblasts isolated from normal subjects. As expected, NPC1 mutant cells displayed an inappropriate homeostatic response to accumulated intracellular cholesterol. In addition, a number of striking parallels were observed between NPC disease and Alzheimer's disease.Many genes involved in the trafficking and processing of amyloid precursor protein and the microtubule binding protein, tau, were more highly expressed. Numerous genes important for membrane traffic and the cellular regulation of calcium, metals and other ions were upregulated. Finally, NPC fibroblasts exhibited a gene expression profile indicative of oxidative stress. These changes are likely contributors to the pathophysiology of Niemann-Pick Type C disease.

  View details for DOI 10.1371/journal.pone.0000019

  View details for Web of Science ID 000207443600019

  View details for PubMedID 17183645

  View details for PubMedCentralID PMC1762405

• A functional role for the GCC185 Golgin in mannose 6-phosphate receptor recycling MOLECULAR BIOLOGY OF THE CELL Reddy, J. V., Burguete, A. S., Sridevi, K., Ganley, I. G., Nottingham, R. M., Pfeffer, S. R. 2006; 17 (10): 4353-4363

  Abstract

  Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.

  View details for DOI 10.1091/mbc.E06-02-0153

  View details for Web of Science ID 000241087300018

  View details for PubMedID 16885419

  View details for PubMedCentralID PMC1635343

• Lab management: insights for the new investigator NATURE IMMUNOLOGY Haynes, L., Pfeffer, S., Boss, J. M., Kavathas, P., Kuchroo, V. 2006; 7 (9): 895-897

  Abstract

  A well functioning lab is a productive lab. Here the guiding principles of good lab management are discussed.

  View details for DOI 10.1038/ni0906-895

  View details for Web of Science ID 000239942300003

  View details for PubMedID 16924248

• Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells JOURNAL OF BIOLOGICAL CHEMISTRY Ganley, I. G., Pfeffer, S. R. 2006; 281 (26): 17890-17899

  Abstract

  Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.

  View details for DOI 10.1074/jbc.M601679200

  View details for Web of Science ID 000238490300039

  View details for PubMedID 16644737

  View details for PubMedCentralID PMC3650718

• TIP47 is a key effector for Rab9 localization JOURNAL OF CELL BIOLOGY Aivazian, D., Serrano, R. L., Pfeffer, S. 2006; 173 (6): 917-926

  Abstract

  The human genome encodes approximately 70 Rab GTPases that localize to the surfaces of distinct membrane compartments. To investigate the mechanism of Rab localization, chimeras containing heterologous Rab hypervariable domains were generated, and their ability to bind seven Rab effectors was quantified. Two chimeras could bind effectors for two distinctly localized Rabs; a Rab5/9 hybrid bound both Rab5 and Rab9 effectors, and a Rab1/9 hybrid bound to certain Rab1 and Rab9 effectors. These unusual chimeras permitted a test of the importance of effector binding for Rab localization. In both cases, changing the cellular concentration of a key Rab9 effector, which is called tail-interacting protein of 47 kD, moved a fraction of the proteins from their parental Rab localization to that of Rab9. Thus, relative concentrations of certain competing effectors could determine a chimera's localization. These data confirm the importance of effector interactions for Rab9 localization, and support a model in which effector proteins rely on Rabs as much as Rabs rely on effectors to achieve their correct steady state localizations.

  View details for Web of Science ID 000238585700011

  View details for PubMedID 16769818

• Misincorporation proton-alkyl exchange (MPAX): engineering cysteine probes into proteins. Current protocols in protein science / editorial board, John E. Coligan ... [et al.] Burguete, A. S., Harbury, P. B., Pfeffer, S. R. 2005; Chapter 26: Unit26 1-?

  Abstract

  This unit describes a rapid and efficient method to screen a polypeptide for amino acid residues that contribute to protein-protein interaction interfaces. Cysteine residues are introduced as positional probes in a protein at random by co-expression in bacteria with specific cysteine misincorporator tRNAs. The protein is then purified as an ensemble of polypeptides containing cysteine at low frequency, at different positions in each molecule. The ability of the native protein structure to protect different cysteine residues from chemical modification by iodoacetamide is determined to obtain a protein surface map that reveals candidate surface residues that are likely to be important for protein-protein interaction. Cysteine mutants with altered ligand binding can also be selected simultaneously by affinity chromatography.

  View details for DOI 10.1002/0471140864.ps2601s42

  View details for PubMedID 18429287

• Filling the Rab GAP NATURE CELL BIOLOGY Pfeffer, S. 2005; 7 (9): 856-857

  View details for DOI 10.1038/ncb0905-856

  View details for Web of Science ID 000231601600007

  View details for PubMedID 16136184

• A model for rab GTPase localization Conference on Localization and Activation of Ras-Like GTPases Pfeffer, S. PORTLAND PRESS LTD. 2005: 627–630

  Abstract

  The human genome encodes almost 70 Rab GTPases. These proteins are C-terminally geranylgeranylated and are localized to the surfaces of distinct membrane-bound compartments in eukaryotic cells. This mini review presents a working model for how Rabs achieve and maintain their steady-state localizations. Data from a number of laboratories suggest that Rabs participate in the generation of macromolecular assemblies that generate functional microdomains within a given membrane compartment. Our data suggest that these complex interactions are important for the cellular localization of Rab proteins at steady state.

  View details for Web of Science ID 000231345700020

  View details for PubMedID 16042559

• Structural clues to Rab GTPase functional diversity JOURNAL OF BIOLOGICAL CHEMISTRY Pfeffer, S. R. 2005; 280 (16): 15485-15488

  Abstract

  Rab GTPases are key regulators of membrane trafficking in eukaryotes. Recent structural analysis of a number of Rabs, either alone or in complex with partner proteins, has provided new insight into the importance of both conserved and non-conserved features of these proteins that specify their unique functions and localizations. This review will highlight what we have learned from crystallographic analysis of this important protein family.

  View details for DOI 10.1074/jbc.R500003200

  View details for Web of Science ID 000228444800003

  View details for PubMedID 15746102

• Purification and properties of Yip3/PRA1 as a Rab GDI displacement factor GTPASES REGULATING MEMBRANE TARGETING AND FUSION Sivars, U., Aivazian, D., Pfeffer, S. 2005; 403: 348-356

  Abstract

  Prenylated Rab proteins exist in the cytosol bound to guanine dissociation inhibitor (GDI). These dimeric complexes contain all of the information needed for accurate membrane delivery. We have shown that membranes contain a proteinaceous activity that is required for Rab delivery, and we named that activity GDI displacement factor (GDF). Biochemical analysis revealed that GDF activity was membrane associated and had a mass of approximately 25 kDa. We therefore used a candidate gene approach and were able to show that pure Yip3/PRA1 protein displays GDF activity. In this chapter, we review key aspects of GDF analysis: our assay and the method by which we purify Yip3/PRA1 in active form.

  View details for DOI 10.1016/S0076-6879(05)03030-2

  View details for Web of Science ID 000234855400030

  View details for PubMedID 16473601

• Purification and analysis of TIP47 function in Rab9-dependent mannose 6-phosphate receptor trafficking GTPASES REGULATING MEMBRANE TARGETING AND FUSION Burguete, A. S., Sivars, U., Pfeffer, S. 2005; 403: 357-366

  Abstract

  TIP47 (tail interacting protein of 47 kDa) is a cytosolic protein that is essential for the transport of mannose 6-phosphate receptors (MPRs) from endosomes to the trans-Golgi. This protein is recruited from the cytosol onto the surface of late endosomes by Rab9 GTPase, which enables TIP47 to bind to MPR cytoplasmic domains with enhanced affinity. A mutation in a deep hydrophobic cleft of TIP47 (F(236)C) confers enhanced affinity binding to MPR cytoplasmic domains and stabilizes MPRs in living cells. We describe the purification of native and recombinant TIP47 proteins and assays that we use to monitor the function of this protein in MPR transport in living cells.

  View details for Web of Science ID 000234855400031

  View details for PubMedID 16473602

• Rab9 GTPase regulates late endosome size and requires effector interaction for its stability MOLECULAR BIOLOGY OF THE CELL Ganley, I. G., CARROLL, K., Bittova, L., Pfeffer, S. 2004; 15 (12): 5420-5430

  Abstract

  Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.

  View details for DOI 10.1091/mbc.E04-08-0747

  View details for Web of Science ID 000225372800019

  View details for PubMedID 15456905

  View details for PubMedCentralID PMC532021

• Targeting RAB GTPases to distinct membrane compartments NATURE REVIEWS MOLECULAR CELL BIOLOGY Pfeffer, S., Aivazian, D. 2004; 5 (11): 886-896

  Abstract

  Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments. The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery. The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.

  View details for DOI 10.1038/nrm1500

  View details for Web of Science ID 000224987300013

  View details for PubMedID 15520808

• In vitro selection and prediction of TIP47 protein-interaction interfaces NATURE METHODS Burguete, A. S., Harbury, P. B., Pfeffer, S. R. 2004; 1 (1): 55-60

  Abstract

  We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.

  View details for DOI 10.1038/NMETH702

  View details for Web of Science ID 000226753700019

  View details for PubMedID 15782153

• Yip3 catalyses the dissociation of endosomal Rab-GDI complexes NATURE Sivars, U., Aivazian, D., Pfeffer, S. R. 2003; 425 (6960): 856-859

  Abstract

  Human cells contain more than 60 small G proteins of the Rab family, which are localized to the surfaces of distinct membrane compartments and regulate transport vesicle formation, motility, docking and fusion. Prenylated Rabs also occur in the cytosol bound to GDI (guanine nucleotide dissociation inhibitor), which binds to Rabs in their inactive state. Prenyl Rab-GDI complexes contain all of the information necessary to direct Rab delivery onto distinct membrane compartments. The late endosomal, prenyl Rab9 binds GDI with very high affinity, which led us to propose that there might be a 'GDI-displacement factor' to catalyse dissociation of Rab-GDI complexes and to enable transfer of Rabs from GDI onto membranes. Indeed, we have previously shown that endosomal membranes contain a proteinaceous factor that can act in this manner. Here we show that the integral membrane protein, Yip3, acts catalytically to dissociate complexes of endosomal Rabs bound to GDI, and to deliver them onto membranes. We propose that the conserved Yip proteins serve as GDI-displacement factors for the targeting of Rab GTPases in eukaryotic cells.

  View details for DOI 10.1038/nature02057

  View details for Web of Science ID 000186118500049

  View details for PubMedID 14574414

• Membrane domains in the secretory and endocytic pathways CELL Pfeffer, S. 2003; 112 (4): 507-517

  Abstract

  Progress in identifying, characterizing, and localizing the constituents of distinct membrane bound compartments has revealed a new level of intracellular subcompartmentation. Proteins and lipids are not uniformly distributed in a given organelle, and subdomains are formed by a combination of hierarchical assembly processes and protein exclusion. Thus, functionally distinct specializations of a given organelle are physically segregated to a greater extent than previously believed.

  View details for Web of Science ID 000181252600009

  View details for PubMedID 12600314

• Self-assembly is important for TIP47 function in mannose 6-phosphate receptor transport TRAFFIC Sincock, P. M., Ganley, I. G., Krise, J. P., Diederichs, S., Sivars, U., O'Connor, B., Ding, L., Pfeffer, S. R. 2003; 4 (1): 18-25

  Abstract

  TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the trans-Golgi network in vitro and in living cells. TIP47 occurs in cytosol as an oligomer; it chromatographs with an apparent mass of approximately 300 kDa and displays an S-value of approximately 13. Recombinant TIP47 forms homo-oligomers that are likely to represent hexamers, as determined by chemical cross-linking. Removal of TIP47 residues 1-151 yields a protein that behaves as a monomer upon gel filtration, yet is fully capable of binding mannose 6-phosphate receptor cytoplasmic domains. The presence of an oligomerization domain in the N-terminus of TIP47 was confirmed by expression of N-terminal residues 1-133 or 1-257 in mammalian cells. Co-expression of full-length TIP47 with either of these fragments led to the formation of higher-order aggregates of wild-type TIP47. Furthermore, the N-terminal domains expressed alone also occurred as oligomers. These studies reveal an N-terminal oligomerization domain in TIP47, and show that oligomerization is not required for TIP47 recognition of mannose 6-phosphate receptors. However, oligomerization is required for TIP47 stimulation of mannose 6-phosphate receptor transport from endosomes to the trans-Golgi in vivo.

  View details for Web of Science ID 000180423300004

  View details for PubMedID 12535272

• Identification of residues in TIP47 essential for Rab9 binding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hanna, J., CARROLL, K., Pfeffer, S. R. 2002; 99 (11): 7450-7454

  Abstract

  TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-dependent and cation-independent mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the trans-Golgi network in vitro and in living cells. TIP47 recognizes distinct determinants in the cytoplasmic domains of these two receptors, and its ability to bind to the cation-independent MPR is enhanced by the concomitant binding of the Rab9 GTPase. We show here that TIP47 residues 161-169 are essential, but likely not sufficient, for Rab9 binding. Mutation of these residues led to a significant decrease in Rab9 binding, but did not alter the global folding of the protein. The most impaired mutant was indistinguishable from wild-type TIP47 in its circular dichroism spectrum, and mutant proteins that showed decreased Rab9 binding retained full capacity to bind to MPR cytoplasmic domains. Closely related sequences in a related protein, adipophilin, did not confer Rab9 binding capacity to that protein. Partial proteolysis of TIP47 and TIP47 mutant proteins revealed subtle conformational differences, suggesting that residues 161-169 reside in a portion of TIP47 that is important for its conformation. These experiments reveal distinct binding domains for the Rab9 GTPase and MPR cytoplasmic domains in the cargo selection protein TIP47.

  View details for DOI 10.1073/pnas.112198799

  View details for Web of Science ID 000175908600025

  View details for PubMedID 12032303

  View details for PubMedCentralID PMC124251

• Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells JOURNAL OF CELL BIOLOGY Barbero, P., Bittova, L., Pfeffer, S. R. 2002; 156 (3): 511-518

  Abstract

  Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

  View details for DOI 10.1083/jcb.200109030

  View details for Web of Science ID 000176425500009

  View details for PubMedID 11827983

  View details for PubMedCentralID PMC2173336

• Constructing a Golgi complex JOURNAL OF CELL BIOLOGY Pfeffer, S. R. 2001; 155 (6): 873-875

  Abstract

  In this issue, Short et al. report the discovery of a protein named Golgin-45 that is located on the surface of the middle (or medial) cisternae of the Golgi complex. Depletion of this protein disrupts the Golgi complex and leads to the return of a resident, lumenal, medial Golgi enzyme to the endoplasmic reticulum. These findings suggest that Golgin-45 serves as a linchpin for the maintenance of Golgi complex structure, and offer hints as to the mechanisms by which the polarized Golgi complex is constructed.

  View details for Web of Science ID 000172730200001

  View details for PubMedID 11739400

• Rab GTPases: specifying and deciphering organelle identity and function TRENDS IN CELL BIOLOGY Pfeffer, S. R. 2001; 11 (12): 487-491

  Abstract

  Ten years ago, 20 Rab proteins had been identified as organelle-specific GTPases, and two were known to be essential for vesicle targeting in yeast. Today, more than 60 mammalian Rab proteins have been identified. While Rabs were always viewed as key regulatory factors, no one could have anticipated their diversity of functions and multitude of effectors. Rabs organize distinct protein scaffolds within a single organelle and act in a combinatorial manner with their effectors to regulate all stages of membrane traffic.

  View details for Web of Science ID 000172530100011

  View details for PubMedID 11719054

• Vesicle tethering factors united MOLECULAR CELL Pfeffer, S. 2001; 8 (4): 729-730

  Abstract

  In the October 2001 issue of Developmental Cell, Whyte and Munro elucidate the composition of a novel vesicle tethering complex and in the process uncover previously undetected homology between tethering complexes that catalyze a variety of different transport events in yeast and mammalian cells.

  View details for Web of Science ID 000171908900001

  View details for PubMedID 11684005

• TIP47 is not a component of lipid droplets JOURNAL OF BIOLOGICAL CHEMISTRY Barbero, P., Buell, E., Zulley, S., Pfeffer, S. R. 2001; 276 (26): 24348-24351

  Abstract

  TIP47 functions in the delivery of mannose 6-phosphate receptors from endosomes to the trans-Golgi network both in vitro and in vivo. It binds directly and very specifically to the cytoplasmic domains of both the cation-independent and cation-dependent mannose 6-phosphate receptors. TIP47 is 43% identical to a lipid droplet-associated protein named adipophilin; much of the identity resides near the N termini of these proteins. It was recently reported in this journal, in a study using antiserum from this laboratory, that TIP47 is a constituent of lipid droplets (Wolins, N. E., Rubin, B., and Brasaemle, D. L. (2001) J. Biol. Chem. 276, 5101-5108). We show here that the findings of Wolins et al. were likely due to either a cross-reactive, unidentified protein in HeLa cells that is recognized by our antiserum and/or the fact that our serum also cross-reacts with the adipophilin protein itself, shown directly by expression of adipophilin in Escherichia coli. Using antibodies specific for residues 152-434 of TIP47, we show that TIP47 is not a constituent of lipid droplets.

  View details for Web of Science ID 000169531100147

  View details for PubMedID 11313361

• Role of Rab9 GTPase in facilitating receptor recruitment by TIP47 SCIENCE Carroll, K. S., Hanna, J., Simon, I., Krise, J., Barbero, P., Pfeffer, S. R. 2001; 292 (5520): 1373-1376

  Abstract

  Mannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases from the Golgi to endosomes and then return to the Golgi complex. TIP47 recognizes the cytoplasmic domains of MPRs and is required for endosome-to-Golgi transport. Here we show that TIP47 also bound directly to the Rab9 guanosine triphosphatase (GTPase) in its active, GTP-bound conformation. Moreover, Rab9 increased the affinity of TIP47 for its cargo. A functional Rab9 binding site was required for TIP47 stimulation of MPR transport in vivo. Thus, a cytosolic cargo selection device may be selectively recruited onto a specific organelle, and vesicle budding might be coupled to the presence of an active Rab GTPase.

  View details for Web of Science ID 000168862900045

  View details for PubMedID 11359012

• Caveolae on the move NATURE CELL BIOLOGY Pfeffer, S. R. 2001; 3 (5): E108-E110

  View details for Web of Science ID 000168592500006

  View details for PubMedID 11331891

• Membrane transport: Retromer to the rescue CURRENT BIOLOGY Pfeffer, S. R. 2001; 11 (3): R109-R111

  Abstract

  Genetic analysis in yeast has led to the discovery of a complex that retrieves proteins selectively from the prevacuolar compartment and transports them to the Golgi. Orthologs of these proteins in mammalian cells are likely to play a similar role but their cargoes are yet to be identified.

  View details for Web of Science ID 000169076300012

  View details for PubMedID 11231171

• Quantitative analysis of TIP47-receptor cytoplasmic domain interactions - Implications for endosome-to-trans Golgi network trafficking JOURNAL OF BIOLOGICAL CHEMISTRY Krise, J. P., Sincock, P. M., Orsel, J. G., Pfeffer, S. R. 2000; 275 (33): 25188-25193

  Abstract

  TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-independent and cation-dependent mannose 6-phosphate receptors and is required for their transport from late endosomes to the trans Golgi network in vitro and in vivo. We report here a quantitative analysis of the interaction of recombinant TIP47 with mannose 6-phosphate receptor cytoplasmic domains. Recombinant TIP47 binds more tightly to the cation-independent mannose 6-phosphate receptor (K(D) = 1 microm) than to the cation-dependent mannose 6-phosphate receptor (K(D) = 3 microm). In addition, TIP47 fails to interact with the cytoplasmic domains of the hormone-processing enzymes, furin, phosphorylated furin, and metallocarboxypeptidase D, as well as the cytoplasmic domain of TGN38, proteins that are also transported from endosomes to the trans Golgi network. Although these proteins failed to bind TIP47, furin and TGN38 were readily recognized by the clathrin adaptor, AP-2. These data suggest that TIP47 recognizes a very select set of cargo molecules. Moreover, our data suggest unexpectedly that furin, TGN38, and carboxypeptidase D may use a distinct vesicular carrier and perhaps a distinct route for transport between endosomes and the trans Golgi network.

  View details for Web of Science ID 000088849400021

  View details for PubMedID 10829017

• Recognition of the 300-kDa mannose 6-phosphate receptor cytoplasmic domain by 47-kDa tail-interacting protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Orsel, J. G., Sincock, P. M., Krise, J. P., Pfeffer, S. R. 2000; 97 (16): 9047-9051

  Abstract

  Tail-interacting 47-kDa protein (TIP47) binds the cytoplasmic domains of the cation-dependent (CD) and cation-independent (CI) mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the Golgi complex. TIP47 recognizes a phenylalanine-tryptophan signal in the CD-MPR. We show here that TIP47 interaction with the 163-residue CI-MPR cytoplasmic domain is highly conformation dependent and requires CI-MPR residues that are proximal to the membrane. CI-MPR cytoplasmic domain residues 1-47 are dispensable, whereas residues 48-74 are essential for high-affinity binding. However, residues 48-74 are not sufficient for high-affinity binding; residues 75-163 alone display weak affinity for TIP47, yet they contribute to the presentation of residues 48-74 in the intact protein. Independent competition binding experiments confirm that TIP47 interacts with the membrane-proximal portion of the CI-MPR cytoplasmic domain. TIP47 binding is competed by the binding of the AP-2 clathrin adaptor at (and near) residues 24-29 but not by AP-1 binding at (and near) residues 160-161. Finally, TIP47 appears to recognize a putative loop generated by the sequence PPAPRPG and other hydrophobic residues in the membrane-proximal domain. Although crystallography will be needed to define the precise interaction interface, these data provide an initial structural basis for TIP47-CI-MPR association.

  View details for Web of Science ID 000088608000053

  View details for PubMedID 10908666

• Recognition of the cation-independent mannose 6-phosphate receptor by TIP47. Orsel, J. G., Krise, J. P., Diaz, E., Pfeffer AMER SOC CELL BIOLOGY. 1999: 224A

  View details for Web of Science ID 000083673501302

• Motivating endosome motility NATURE CELL BIOLOGY Pfeffer, S. R. 1999; 1 (6): E145-E147

  View details for Web of Science ID 000083169800005

  View details for PubMedID 10559973

• A retroviral gene trap insertion at the mouse rab9 locus. Cannon, J. P., Colicos, S. M., Pfeffer, Belmont, J. W. UNIV CHICAGO PRESS. 1999: A232

  View details for Web of Science ID 000082879801287

• Membrane tethering in intracellular transport CURRENT OPINION IN CELL BIOLOGY Waters, M. G., Pfeffer, S. R. 1999; 11 (4): 453-459

  Abstract

  Studies of various membrane trafficking steps over the past year indicate that membranes are tethered together prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction. The tethering proteins identified to date are quite large, being either fibrous proteins or multimeric protein complexes. The tethering factors employed at different steps are evolutionarily unrelated, yet their function seems to be closely tied to the more highly conserved Rab GTPases. Tethering factors may collaborate with Rabs and SNAREs to generate targeting specificity in the secretory pathway.

  View details for Web of Science ID 000082107400006

  View details for PubMedID 10449330

• Mapmodulin, cytoplasmic dynein, and microtubules enhance the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network MOLECULAR BIOLOGY OF THE CELL Itin, C., Ulitzur, N., Muhlbauer, B., Pfeffer, S. R. 1999; 10 (7): 2191-2197

  Abstract

  Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

  View details for Web of Science ID 000081515400008

  View details for PubMedID 10397758

• Transport-vesicle targeting: tethers before SNAREs NATURE CELL BIOLOGY Pfeffer, S. R. 1999; 1 (1): E17-E22

  Abstract

  Protein secretion and the transport of proteins between membrane-bound compartments are mediated by small, membrane-bound vesicles. Here I review what is known about the process by which vesicles are targeted to the correct destination. A growing family of proteins, whose precise modes of action are far from established, is involved in targeting. Despite the wide diversity in the identities of the players, some common themes are emerging that may explain how vesicles can identify their targets and release their cargo at the correct time and place in eukaryotic cells.

  View details for Web of Science ID 000083085300009

  View details for PubMedID 10559876

• Molecular analysis of receptor trafficking Pfeffer, Diaz, E., Orsel, J., Krise, J., Simon FEDERATION AMER SOC EXP BIOL. 1999: A1428

  View details for Web of Science ID 000082033400611

• TIP47, a cargo selection device for mannose 6-phosphate receptor trafficking Diaz, E., Schimmoller, F., Orsel, J., Pfeffer AMER SOC CELL BIOLOGY. 1998: 471A

  View details for Web of Science ID 000076906702731

• Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution GENE Schimmoller, F., Diaz, E., Muhlbauer, B., Pfeffer, S. R. 1998; 216 (2): 311-318

  Abstract

  We report here the identification and characterization of a human 76kDa membrane protein that is found predominantly in endosomes. This protein is related to the Saccharomyces cerevisiae EMP70 gene product, a precursor protein whose 24kDa cleavage product (p24a) was found in yeast endosome-enriched membrane fractions (Singer-Krüger et al., 1993. J. Biol. Chem. 268, 14376-14386). Northern blot analysis indicated that p76 mRNA is highly expressed in human pancreas but could be detected in all tissues examined. p76 is highly conserved throughout evolution, as related proteins have also been detected in Caenorhabditis elegans and Arabidopsis thaliana. This family of proteins has a relatively divergent, hydrophilic N-terminal domain and a well-conserved, highly hydrophobic C-terminal domain which contains nine potential membrane-spanning domains. Transiently expressed, myc-tagged human p76 appears to be localized to endosomes by virtue of its apparent colocalization with transferrin receptors and some mannose 6-phosphate receptors. Furthermore, p76 adopts a type-I topology within the membrane, with its hydrophilic N-terminus facing the lumen of cytoplasmic membranes. The structural features of p76 suggest that it may function as a channel or small molecule transporter in intracellular compartments throughout phylogeny. 1998 Elsevier Science B.V.

  View details for Web of Science ID 000075796500010

  View details for PubMedID 9729438

• Rab GTPases, directors of vesicle docking JOURNAL OF BIOLOGICAL CHEMISTRY Schimmoller, F., Simon, I., Pfeffer, S. R. 1998; 273 (35): 22161-22164

  View details for Web of Science ID 000075616600001

  View details for PubMedID 9712825

• TIP47: A cargo selection device for mannose 6-phosphate receptor trafficking CELL Diaz, E., Pfeffer, S. R. 1998; 93 (3): 433-443

  Abstract

  Mannose 6-phosphate receptors (MPRs) transport newly synthesized lysosomal hydrolases from the Golgi to prelysosomes and then return to the Golgi for another round of transport. We have identified a 47 kDa protein (TIP47) that binds selectively to the cytoplasmic domains of cation-independent and cation-dependent MPRs. TIP47 is present in cytosol and on endosomes and is required for MPR transport from endosomes to the trans-Golgi network in vitro and in vivo. TIP47 recognizes a phenylalanine/tryptophan signal in the tail of the cation-dependent MPR that is essential for its proper sorting within the endosomal pathway. These data suggest that TIP47 binds MPR cytoplasmic domains and facilitates their collection into transport vesicles destined for the Golgi.

  View details for Web of Science ID 000073471500018

  View details for PubMedID 9590177

• Biochemical characterization of mapmodulin, a protein that binds microtubule-associated proteins JOURNAL OF BIOLOGICAL CHEMISTRY Ulitzur, N., RANCANO, C., Pfeffer, S. R. 1997; 272 (48): 30577-30582

  Abstract

  Mapmodulin is a 31-kDa protein that stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. We have shown previously that it binds the microtubule binding domains of the microtubule-associated proteins, MAP2, MAP4, and tau. We also showed that mapmodulin is identical to a protein named PHAPI (Vaesen, M., Barnikol-Watanabe, S. , Götz, H., Awni, L.A., Cole, T., Zimmermann, B., Kratzin, H.D. and Hilschmann, N. (1994) Biol. Chem. Hoppe-Seyler 375, 113-126). We report here that mapmodulin is a phosphoprotein that is predominantly cytosolic but is also found peripherally associated with endoplasmic reticulum and Golgi membranes in mammalian cells. The protein occurs as a trimer in cytosol, and phosphorylation is required for its microtubule-associated protein-binding activity. Heat treatment of nonphosphorylated mapmodulin can render it competent for binding to microtubule-associated proteins, suggesting that phosphorylation induces a conformational change in mapmodulin. Finally, despite identity in polypeptide sequence with a protein reported to act as an inhibitor of protein phosphatase 2A, native mapmodulin was not able to inhibit protein phosphatase 2A in Chinese hamster ovary cell cytosol.

  View details for Web of Science ID A1997YH61300086

  View details for PubMedID 9374554

• A novel assay reveals a role for soluble N-ethylmaleimide-sensitive fusion attachment protein in mannose 6-phosphate receptor transport from endosomes to the trans Golgi network JOURNAL OF BIOLOGICAL CHEMISTRY Itin, C., RANCANO, C., Nakajima, Y., Pfeffer, S. R. 1997; 272 (44): 27737-27744

  Abstract

  Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.

  View details for Web of Science ID A1997YD47300042

  View details for PubMedID 9346916

• Rab GTPases in vesicular transport Pfeffer FEDERATION AMER SOC EXP BIOL. 1997: A1282

  View details for Web of Science ID 000073305602973

• A novel Rab9 effector required for endosome-to-TGN transport JOURNAL OF CELL BIOLOGY Diaz, E., Schimmoller, F., Pfeffer, S. R. 1997; 138 (2): 283-290

  Abstract

  Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (approximately 30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

  View details for Web of Science ID A1997XP26700006

  View details for PubMedID 9230071

• Mapmodulin: A possible modulator of the interaction of microtubule-associated proteins with microtubules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ulitzur, N., Humbert, M., Pfeffer, S. R. 1997; 94 (10): 5084-5089

  Abstract

  We have purified and characterized a 31-kDa protein named mapmodulin that binds to the microtubule-associated proteins (MAPs) MAP2, MAP4, and tau. Mapmodulin binds free MAPs in strong preference to microtubule-associated MAPs, and appears to do so via the MAP's tubulin-binding domain. Mapmodulin inhibits the initial rate of MAP2 binding to microtubules, a property that may allow mapmodulin to displace MAPs from the path of organelles translocating along microtubules. In support of this possibility, mapmodulin stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact CHO cells. To our knowledge, mapmodulin represents the first example of a protein that can bind and potentially regulate multiple MAP proteins.

  View details for Web of Science ID A1997WZ25700049

  View details for PubMedID 9144194

• Vesicle traffic: Get your coat! CURRENT BIOLOGY Schimmoller, F., Itin, C., Pfeffer, S. 1997; 7 (4): R235-R237

  Abstract

  The budding of transport vesicles from the Golgi complex is initiated by activation of the small GTPase ARF; the discovery of enzymes that can convert soluble ARF-GDP to the active, membrane-associated form ARF-GTP will shed light on the mechanism and regulation of the formation of transport vesicles.

  View details for Web of Science ID A1997WV36600012

  View details for PubMedID 9162499

• Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network MOLECULAR BIOLOGY OF THE CELL Nakajima, Y., Pfeffer, S. R. 1997; 8 (4): 577-582

  Abstract

  Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.

  View details for Web of Science ID A1997WU80300004

  View details for PubMedID 9247639

• Identification of a GDI displacement factor that releases endosomal Rab GTPases from Rab-GDI EMBO JOURNAL DIRACSVEJSTRUP, A. B., Sumizawa, T., Pfeffer, S. R. 1997; 16 (3): 465-472

  Abstract

  Prenylated Rab GTPases occur in the cytosol in their GDP-bound conformations bound to a cytosolic protein termed GDP-dissociation inhibitor (GDI). Rab-GDI complexes represent a pool of active, recycling Rab proteins that can deliver Rabs to specific and distinct membrane-bound compartments. Rab delivery to cellular membranes involves release of GDI, and the membrane-associated Rab protein then exchanges its bound GDP for GTP. We report here the identification of a novel, membrane-associated protein factor that can release prenylated Rab proteins from GDI. This GDI-displacement factor (GDF) is not a guanine nucleotide exchange factor because it did not influence the intrinsic rates of nucleotide exchange by Rabs 5, 7 or 9. Rather, GDF caused the release of each of these endosomal Rabs from GDI, permitting them to exchange nucleotide at their intrinsic rates. GDF displayed the greatest catalytic rate enhancement on Rab9-GDI complexes. However, catalytic rate enhancement paralleled the potency of GDI in blocking nucleotide exchange: GDI was shown to be most potent in blocking nucleotide exchange by Rab9. The failure of GDF to act on Rab1-GDI complexes suggests that it may be specific for endosomal Rab proteins. This novel, membrane-associated activity may be part of the machinery used to localize Rabs to their correct intracellular compartments.

  View details for Web of Science ID A1997WH23600003

  View details for PubMedID 9034329

• Transport vesicle docking: SNAREs and associates ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Pfeffer, S. R. 1996; 12: 441-461

  Abstract

  Proteins that function in transport vesicle docking are being identified at a rapid rate. So-called v- and t-SNAREs form the core of a vesicle docking complex. Additional accessory proteins are required to protect SNAREs from promiscuous binding and to deprotect SNAREs under conditions in which transport vesicle docking should occur. Because access to SNAREs must be regulated, other proteins must also contain specificity determinants to accomplish delivery of transport vesicles to their distinct and specific membrane targets.

  View details for Web of Science ID A1996VY42800016

  View details for PubMedID 8970734

• RAB7 AND RAB9 ARE RECRUITED ONTO LATE ENDOSOMES BY BIOCHEMICALLY DISTINGUISHABLE PROCESSES JOURNAL OF BIOLOGICAL CHEMISTRY Soldati, T., RANCANO, C., Geissler, H., Pfeffer, S. R. 1995; 270 (43): 25541-25548

  Abstract

  Rab GTPases are localized to the surfaces of distinct membrane-bound organelles and function in transport vesicle docking and/or fusion. Prenylated Rab9, bound to GDP dissociation inhibitor-alpha, can be recruited selectively onto a membrane fraction enriched in late endosomes; this process is accompanied by nucleotide exchange. We used this system to address whether each Rab uses a distinct machinery to associate with its cognate organelle. Purified, prenylated Rab1B, Rab7, and Rab9 proteins were each reconstituted as stoichiometric complexes with purified GDP dissociation inhibitor-alpha, and their recruitment onto endosome- or ER-enriched membrane fractions was quantified. The two late endosomal proteins, Rab9 and Rab7, were each recruited onto endosome membranes with approximate apparent Km values of 9 and 22 nM, respectively. However, while control Rab9.GDP dissociation inhibitor-alpha complexes inhibited the initial rate of myc-tagged Rab9 recruitment with an apparent Ki of approximately 9 nM, Rab7 complexes inhibited this process much less effectively (apparent Ki approximately 112 nM). Similarly, complexes of the endoplasmic reticulum-localized Rab1B protein were even less potent than Rab7 complexes (apparent Ki approximately 405 nm). Rab9 complexes inhibited Rab7 recruitment with the same low efficacy as Rab7 complexes inhibited Rab9 recruitment. These experiments distinguish, biochemically, the recruitment of different Rab proteins onto a single class of organelle. Since Rab7 and Rab9 are both localized at least in large part, to late endosomes, this suggests that a single organelle may bear multiple Rab recruitment machines.

  View details for Web of Science ID A1995TB46600041

  View details for PubMedID 7592724

• RAB-GDP DISSOCIATION INHIBITOR - PUTTING RAB-GTPASES IN THE RIGHT PLACE JOURNAL OF BIOLOGICAL CHEMISTRY Pfeffer, S. R., DIRACSVEJSTRUP, A. B., Soldati, T. 1995; 270 (29): 17057-17059

  View details for Web of Science ID A1995RK68900001

  View details for PubMedID 7615494

• QUANTITATIVE-ANALYSIS OF THE INTERACTIONS BETWEEN PRENYL RAB9, GDP DISSOCIATION INHIBITOR-ALPHA, AND GUANINE-NUCLEOTIDES JOURNAL OF BIOLOGICAL CHEMISTRY Shapiro, A. D., Pfeffer, S. R. 1995; 270 (19): 11085-11090

  Abstract

  Rab9 is a Ras-like GTPase required for the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Rab9 occurs in the cytosol as a complex with GDP dissociation inhibitor (GDI), which we have shown delivers prenyl Rab9 to late endosomes in a functional form. We report here basal rate constants for guanine nucleotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate constants were influenced in part by the hydrophobic environment of the prenyl group. Guanine nucleotide dissociation and GTP hydrolysis rates were lower in the presence of lipid; detergent stimulated intrinsic nucleotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a KD of 60 nM in 0.1% Lubrol and 23 nM in 0.02% Lubrol. In 0.1% Lubrol, GDI-alpha inhibited GDP dissociation half maximally at 72 +/- 18 nM, consistent with the KD determinations. These data suggest that GDI-alpha associates with prenyl Rab9 with a KD of < or = 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 1.9-fold and bound prenyl Rab9 with a KD of 67 nM in 0.1% Lubrol.

  View details for Web of Science ID A1995QX86500013

  View details for PubMedID 7744738

• EXPRESSION OF RAB9 PROTEIN IN ESCHERICHIA-COLI - PURIFICATION AND ISOPRENYLATION IN-VITRO SMALL GTPASES AND THEIR REGULATORS, PT C Riederer, M. A., Soldati, T., DIRACSVEJSTRUP, A. B., Pfeffer, S. R. 1995; 257: 15-21

  View details for Web of Science ID A1995BE08F00003

  View details for PubMedID 8583917

• Regulation of receptor trafficking by ras-like GTPases Symposium on Alzheimers Disease - Lessons from Cell Biology, at the 10th Medicine and Research Colloquium of the Fondation IPSEN Devoted to Research on Alzheimers Disease Pfeffer, S. R. SPRINGER-VERLAG BERLIN. 1995: 27–36

  View details for Web of Science ID A1995BF14G00003

• Selective membrane recruitment of Rab GTPases Cold Spring Harbor Symposia on Quantitative Biology - Protein Kinesis: The Dynamics of Protein Trafficking and Stability Pfeffer, S. R., Soldati, T., Geissler, H., RANCANO, C., DiracSvejstrup, B. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1995: 221–227

  View details for Web of Science ID A1995VA12500024

  View details for PubMedID 8824394

• RECONSTITUTION OF RAB9 ENDOSOMAL TARGETING AND NUCLEOTIDE EXCHANGE USING PURIFIED RAB9-GDP DISSOCIATION INHIBITOR COMPLEXES AND ENDOSOME-ENRICHED MEMBRANES SMALL GTPASES AND THEIR REGULATORS, PT C Soldati, T., Shapiro, A. D., Pfeffer, S. R. 1995; 257: 253-259

  View details for Web of Science ID A1995BE08F00028

  View details for PubMedID 8583928

• RAB GTPASES - MASTER REGULATORS OF MEMBRANE TRAFFICKING CURRENT OPINION IN CELL BIOLOGY Pfeffer, S. R. 1994; 6 (4): 522-526

  Abstract

  Rab GTPases are thought to be likely to catalyze the accurate association of pairs of targeting molecules located on the surfaces of transport vesicles with their corresponding membrane acceptors. Advances during the past year have solidified our understanding of the mechanisms by which Rab proteins are recruited onto nascent transport vesicles and retrieved from their fusion targets. Functional analyses of Rab proteins in living cells have led to the surprising observation that vesicles do not seem to form if the appropriate Rab protein, in its GTP-bound conformation, is not present.

  View details for Web of Science ID A1994PA83500005

  View details for PubMedID 7986528

• RAB-GDI PRESENTS FUNCTIONAL RAB9 TO THE INTRACELLULAR-TRANSPORT MACHINERY AND CONTRIBUTES SELECTIVITY TO RAB9 MEMBRANE RECRUITMENT JOURNAL OF BIOLOGICAL CHEMISTRY DIRACSVEJSTRUP, A. B., Soldati, T., Shapiro, A. D., Pfeffer, S. R. 1994; 269 (22): 15427-15430

  Abstract

  Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI). We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro. Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity. Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein. Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting. Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9. These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.

  View details for Web of Science ID A1994NP51300008

  View details for PubMedID 8195183

• MEMBRANE TARGETING OF THE SMALL GTPASE RAB9 IS ACCOMPANIED BY NUCLEOTIDE EXCHANGE NATURE Soldati, T., Shapiro, A. D., SVEJSTRUP, A. B., Pfeffer, S. R. 1994; 369 (6475): 76-78

  Abstract

  The Rab GTPases are key regulators of vesicular transport. A fraction of Rab proteins is present in the cytosol, bound with GDP, complexed to a protein termed GDI. Rab9 is localized primarily to late endosomes, where it aids the transport of mannose 6-phosphate receptors to the trans-Golgi network. It has been proposed that Rab proteins are delivered to specific membranes by GDI, and that this process is accompanied by the exchange of bound GDP for GTP. In addition, Rab localization requires carboxy-terminal prenylation and specific structural determinants. Here we describe the reconstitution of the selective targeting of prenylated Rab9 protein onto late endosome membranes and show that this process is accompanied by endosome-triggered nucleotide exchange.

  View details for Web of Science ID A1994NJ86000057

  View details for PubMedID 8164745

• LYSOSOME BIOGENESIS REQUIRES RAB9 FUNCTION AND RECEPTOR RECYCLING FROM ENDOSOMES TO THE TRANS-GOLGI NETWORK JOURNAL OF CELL BIOLOGY Riederer, M. A., Soldati, T., Shapiro, A. D., Lin, J., Pfeffer, S. R. 1994; 125 (3): 573-582

  Abstract

  Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane-associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.

  View details for Web of Science ID A1994NJ95000006

  View details for PubMedID 7909812

• RECEPTOR EXTRACELLULAR DOMAINS MAY CONTAIN TRAFFICKING INFORMATION - STUDIES OF THE 300-KDA MANNOSE 6-PHOSPHATE RECEPTOR JOURNAL OF BIOLOGICAL CHEMISTRY Dintzis, S. M., Velculescu, V. E., Pfeffer, S. R. 1994; 269 (16): 12159-12166

  Abstract

  The 300-kDa mannose 6-phosphate receptor cycles between the trans Golgi network and late endosomes, and between the plasma membrane and early endosomes, to deliver lysosomal enzymes to prelysosomes. Mannose 6-phosphate receptor trafficking requires structural determinants present in the cytoplasmic domain. However, when this domain was joined with the extracellular and transmembrane domains of the epidermal growth factor receptor, it was not sufficient to direct this chimera to late endosomes and the trans Golgi network (Dintzis, S. M., and Pfeffer, S. R. (1990) EMBO J. 9, 77-84). These findings suggested a role for extracellular and/or transmembrane domains in mannose 6-phosphate receptor trafficking. We describe here the construction and expression of chimeric receptors comprised of mannose 6-phosphate receptor extracellular and transmembrane sequences joined with cytoplasmic domain sequences derived from the human epidermal growth factor receptor or the human low density lipoprotein receptor. The chimeras were stable proteins which were efficiently endocytosed and competent to bind a mannose 6-phosphate-containing ligand. Antibody binding assays and indirect immunofluorescence showed that the chimeras containing the mannose 6-phosphate receptor extracellular domain colocalized with mannose 6-phosphate receptors in intracellular compartments. These experiments suggest that the presence of the mannose 6-phosphate receptor extracellular domain may interfere with the rapid recycling of receptors from early endosomes to the cell surface and detain receptors within endosomes.

  View details for Web of Science ID A1994NG37700074

  View details for PubMedID 8163521

• CLUES TO BRAIN-FUNCTION FROM BAKERS-YEAST PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pfeffer, S. R. 1994; 91 (6): 1987-1988

  View details for Web of Science ID A1994NC04300004

  View details for PubMedID 8134336

• BIOCHEMICAL-ANALYSIS OF RAB9, A RAS-LIKE GTPASE INVOLVED IN PROTEIN-TRANSPORT FROM LATE ENDOSOMES TO THE TRANS GOLGI NETWORK JOURNAL OF BIOLOGICAL CHEMISTRY Shapiro, A. D., Riederer, M. A., Pfeffer, S. R. 1993; 268 (10): 6925-6931

  Abstract

  rab9 is a ras-like GTPase which has been implicated in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. We have expressed recombinant rab9 in Escherichia coli, purified the protein to homogeneity, and initiated a biochemical analysis of this enzyme. rab9 hydrolyzed GTP with a rate constant of 0.0052 min-1 at 37 degrees C. rab7, a highly homologous endosomal GTPase, hydrolyzed GTP with a rate constant of 0.0023 min-1 at 37 degrees C. At this temperature, GDP and GTP each dissociated from rab9 with first-order rate constants of 0.017 min-1. GDP and GTP dissociated from rab7 at 37 degrees C with first-order rate constants of 0.0054 and 0.0024 min-1, respectively. We modified the procedure of John et al. (John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R. (1990) Biochemistry 29, 6058-6065) for the preparation of nucleotide-free ras such that the procedure can now be applied to 1000-fold smaller quantities of protein. Using this method, we prepared microgram quantities of nucleotide-free rab9 in a form which is heat-stable, free of exogenous nucleotide-degrading enzymes and which can be stored at -80 degrees C. At 37 degrees C for GDP and GTP, the second-order rate constants for association with nucleotide-free rab9 were 1.7 x 10(6) M-1 s-1 and 1.2 x 10(5) M-1 s-1, respectively, and equilibrium binding constants were 170 pM and 2.4 nM, respectively.

  View details for Web of Science ID A1993KV14100022

  View details for PubMedID 8463223

• RAB GDI - A SOLUBILIZING AND RECYCLING FACTOR FOR RAB9-PROTEIN MOLECULAR BIOLOGY OF THE CELL Soldati, T., Riederer, M. A., Pfeffer, S. R. 1993; 4 (4): 425-434

  Abstract

  Rab proteins are thought to function in the processes by which transport vesicles identify and/or fuse with their respective target membranes. The bulk of these proteins are membrane associated, but a measurable fraction can be found in the cytosol. The cytosolic forms of rab3A, rab11, and Sec4 occur as equimolar complexes with a class of proteins termed "GDIs," or "GDP dissociation inhibitors." We show here that the cytosolic form of rab9, a protein required for transport between late endosomes and the trans Golgi network, also occurs as a complex with a GDI-like protein, with an apparent mass of approximately 80 kD. Complex formation could be reconstituted in vitro using recombinant rab9 protein, cytosol, ATP, and geranylgeranyl diphosphate, and was shown to require an intact rab9 carboxy terminus, as well as rab9 geranylgeranylation. Monoprenylation was sufficient for complex formation because a mutant rab9 protein bearing the carboxy terminal sequence, CLLL, was prenylated in vitro by geranylgeranyl transferase I and was efficiently incorporated into 80-kD complexes. Purified, prenylated rab9 could also assemble into 80-kD complexes by addition of purified, rab3A GDI. Finally, rab3A-GDI had the capacity to solubilize rab9GDP, but not rab9GTP, from cytoplasmic membranes. These findings support the proposal that GDI proteins serve to recycle rab proteins from their target membranes after completion of a rab protein-mediated, catalytic cycle. Thus GDI proteins have the potential to regulate the availability of specific intracellular transport factors.

  View details for Web of Science ID A1993KY66700007

  View details for PubMedID 8389620

• RAB9 FUNCTIONS IN TRANSPORT BETWEEN LATE ENDOSOMES AND THE TRANS GOLGI NETWORK EMBO JOURNAL Lombardi, D., Soldati, T., Riederer, M. A., Goda, Y., ZERIAL, M., Pfeffer, S. R. 1993; 12 (2): 677-682

  Abstract

  Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.

  View details for Web of Science ID A1993KL71700033

  View details for PubMedID 8440258

  View details for PubMedCentralID PMC413253

• CYTOPLASMIC DYNEIN PARTICIPATES IN THE CENTROSOMAL LOCALIZATION OF THE GOLGI-COMPLEX JOURNAL OF CELL BIOLOGY CORTHESYTHEULAZ, I., Pauloin, A., Pfeffer, S. R. 1992; 118 (6): 1333-1345

  Abstract

  The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.

  View details for Web of Science ID A1992JN94700005

  View details for PubMedID 1387874

• GTP-binding proteins in intracellular transport. Trends in cell biology Pfeffer, S. R. 1992; 2 (2): 41-46

  Abstract

  One of the most exciting recent discoveries in the area of intracellular protein transport is the finding that many organelles involved in exocytic and endocytic membrane traffic have one or more Ras-like GTP-binding proteins on their cytoplasmic face that are specific for each membranous compartment. These proteins are attractive candidates for regulators of transport vesicle formation and the accurate delivery of transport vesicles to their correct targets.

  View details for PubMedID 14731525

• TRANSPORT FROM LATE ENDOSOMES TO TRANS-GOLGI NETWORK IN SEMIINTACT CELL-EXTRACTS METHODS IN ENZYMOLOGY Goda, Y., Soldati, T., Pfeffer, S. R. 1992; 219: 153-159

  View details for Web of Science ID A1992KL57800015

  View details for PubMedID 1487988

• MICROTUBULE-MEDIATED GOLGI CAPTURE BY SEMIINTACT CHINESE-HAMSTER OVARY CELLS METHODS IN ENZYMOLOGY CORTHESYTHEULAZ, I., Pfeffer, S. R. 1992; 219: 159-165

  View details for Web of Science ID A1992KL57800016

  View details for PubMedID 1487989

• MANNOSE-6-PHOSPHATE RECEPTORS AND THEIR ROLE IN PROTEIN SORTING ALONG THE PATHWAY TO LYSOSOMES ANNUAL A N RICHARDS SYMP ON CELLULAR MECHANISMS OF PROTEIN SORTING AND PROCESSING / THE 1990 CONVENTION OF THE PHYSIOLOGICAL SOC OF PHILADELPHIA Pfeffer, S. R. HUMANA PRESS INC. 1991: 131–40

  View details for Web of Science ID A1991KA65600014

  View details for PubMedID 1726883

• IDENTIFICATION OF A NOVEL, N-ETHYLMALEIMIDE-SENSITIVE CYTOSOLIC FACTOR REQUIRED FOR VESICULAR TRANSPORT FROM ENDOSOMES TO THE TRANS-GOLGI NETWORK INVITRO JOURNAL OF CELL BIOLOGY Goda, Y., Pfeffer, S. R. 1991; 112 (5): 823-831

  Abstract

  We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.

  View details for Web of Science ID A1991EZ87600005

  View details for PubMedID 1999460

• Targeting of proteins to the lysosome. Current topics in microbiology and immunology Pfeffer, S. R. 1991; 170: 43-65

  View details for PubMedID 1662126

• COMPARTMENTATION OF THE GOLGI-COMPLEX - BREFELDIN-A DISTINGUISHES TRANS-GOLGI CISTERNAE FROM THE TRANS-GOLGI NETWORK JOURNAL OF CELL BIOLOGY CHEGE, N. W., Pfeffer, S. R. 1990; 111 (3): 893-899

  Abstract

  The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network (TGN). It has recently been reported that the organization of the Golgi complex is disrupted in cells treated with the fungal metabolite, brefeldin-A. Under these conditions, it was shown that resident enzymes of the cis-, medial, and trans-Golgi return to the ER. We report here that 300-kD mannose 6-phosphate receptors, when pulse-labeled within the ER of brefeldin-A-treated cells, acquired numerous N-linked galactose residues with a half time of approximately 2 h, as measured by their ability to bind to RCA-I lectin affinity columns. In contrast, Limax flavus lectin chromatography revealed that less than 10% of these receptors acquired sialic acid after 8 h in brefeldin-A. Two lines of evidence suggested that proteins within and beyond the TGN did not return to the ER in the presence of brefeldin-A. First, the majority of 300-kD mannose 6-phosphate receptors present in the TGN and endosomes did not return to the ER after up to 6 h in brefeldin-A, as determined by their failure to contact galactosyltransferase that had relocated there. Moreover, although mannose 6-phosphate receptors did not acquire sialic acid when present in the ER of brefeldin-A-treated cells, they were readily sialylated when labeled at the cell surface and transported to the TGN. These experiments indicate that galactosyltransferase, a trans-Golgi enzyme, returns to the endoplasmic reticulum in the presence of brefeldin-A, while the bulk of sialyltransferase, a resident of the TGN, does not. Our findings support the proposal that the TGN is a distinct, fourth compartment of the Golgi apparatus that is insensitive to brefeldin-A.

  View details for Web of Science ID A1990DW56900009

  View details for PubMedID 2167898

• ANTIBODIES TO CLATHRIN INHIBIT ENDOCYTOSIS BUT NOT RECYCLING TO THE TRANS GOLGI NETWORK INVITRO SCIENCE Draper, R. K., Goda, Y., Brodsky, F. M., Pfeffer, S. R. 1990; 248 (4962): 1539-1541

  Abstract

  Mannose 6-phosphate receptors carry newly synthesized lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes and then return to the TGN to carry out another round of lysosomal enzyme delivery. Although clathrin-coated vesicles mediate the export of mannose 6-phosphate receptors from the TGN, nothing is known about the transport vesicles used to carry these receptors back to the TGN. Two different in vitro assays used in this study show that an antibody that interferes with clathrin assembly blocks receptor-mediated endocytosis of transferrin, but has no effect on the recycling of the 300-kilodalton mannose 6-phosphate receptor from prelysosomes to the TGN. These results suggest that the transport of mannose 6-phosphate receptors from prelysosomes to the TGN does not involve clathrin.

  View details for Web of Science ID A1990DK40800036

  View details for PubMedID 2163108

• A budding and fusing journey through the secretory pathway. Genetic and In Vitro Analysis of Cell Compartmentalization: A UCLA symposium, Taos, NM, USA, February 3-9, 1990. New biologist Pfeffer, S. R. 1990; 2 (5): 430-434

  Abstract

  Reconstitution of vesicular transport events and the molecular and genetic analysis of the secretory pathway have taken the field of membrane traffic into a new era. Already, proteins have been discovered that facilitate multiple transport steps, and studies of the identities and modes of action of additional transport components, such as those that specify the targets of transport vesicles, will soon follow. Even after we understand how transport vesicles form, find their targets, and then fuse, other fundamental questions will still remain. How are proteins sorted into distinct transport vesicles? How is the directionality of protein transport achieved? How do organelles maintain their identities in the face of large volumes of membrane traffic? Finally, how is membrane traffic regulated? Answers to each of these fundamental questions are likely to be available in the not-too-distant future.

  View details for PubMedID 2126957

• THE MANNOSE 6-PHOSPHATE RECEPTOR CYTOPLASMIC DOMAIN IS NOT SUFFICIENT TO ALTER THE CELLULAR-DISTRIBUTION OF A CHIMERIC EGF RECEPTOR EMBO JOURNAL Dintzis, S. M., Pfeffer, S. R. 1990; 9 (1): 77-84

  Abstract

  Unlike most receptors, 300 kd mannose 6-phosphate receptors (MPRs) are localized primarily in the trans-Golgi network (TGN) and endosomes, and they cycle constitutively between these compartments. Yet, when present at the cell surface, MPRs are internalized together with other cell surface receptors in clathrin-coated vesicles. We constructed a chimeric receptor, comprised of human EGF receptor extracellular and transmembrane domains joined to the bovine MPR cytoplasmic domain, to test whether the MPR cytoplasmic domain contained sufficient information to direct a cell surface receptor into both of these transport pathways. The expressed protein was stable, bound EGF with high affinity, and was efficiently endocytosed and recycled back to the cell surface, in the presence or absence of EGF. If the cytoplasmic domain alone is responsible for sorting native MPRs, chimeric receptors might have been expected to be located primarily in the TGN and in endosomes at steady state. Surprisingly, under conditions in which essentially all endogenous MPRs were intracellular, greater than 85% of the chimeric receptors were located at the cell surface. These experiments demonstrate that the MPR cytoplasmic domain is not sufficient to alter the distribution of the EGF receptor, and suggest a role for extracellular and transmembrane domains in MPR routing.

  View details for Web of Science ID A1990CJ35900010

  View details for PubMedID 2153081

• CELL-FREE SYSTEMS TO STUDY VESICULAR TRANSPORT ALONG THE SECRETORY AND ENDOCYTIC PATHWAYS FASEB JOURNAL Goda, Y., Pfeffer, S. R. 1989; 3 (13): 2488-2495

  Abstract

  Proteins bound for the cell surface, lysosomes, and secretory storage granules share a common pathway of intracellular transport. After their synthesis and translocation into the endoplasmic reticulum, these proteins traverse the secretory pathway by a series of vesicular transfers. Similarly, nutrient and signaling molecules enter cells by endocytosis, and move through the endocytic pathway by passage from one membrane-bound compartment to another. Little is known about the mechanisms by which proteins are collected into transport vesicles, or how these vesicles form, identify their targets, and subsequently fuse with their target membranes. An important advance toward our understanding these processes has come from the establishment of cell-free systems that reconstitute vesicular transfers in vitro. It is now possible to measure, in vitro, the transport of proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport vesicles en route from the trans Golgi network to the cell surface. Along the endocytic pathway, cell-free systems are available to study clathrin-coated vesicle formation, early endosome fusion, and the fusion of late endosomes with lysosomes. Moreover, the selective movement of receptors between late endosomes and the trans Golgi network has also been reconstituted. The molecular mechanisms of vesicular transport are now amenable to elucidation.

  View details for Web of Science ID A1989AY79200005

  View details for PubMedID 2680705

• SELECTIVE RECYCLING OF THE MANNOSE 6-PHOSPHATE IGF-II RECEPTOR TO THE TRANS GOLGI NETWORK INVITRO CELL Goda, Y., Pfeffer, S. R. 1988; 55 (2): 309-320

  Abstract

  Mannose 6-phosphate receptors carry soluble lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes, and then return to the TGN for another round of lysosomal enzyme sorting. We describe here a complementation scheme that detects the vesicular transport of the 300 kd mannose 6-phosphate/IGF-II receptor from prelysosomes to the TGN in cell extracts. In vitro transport displays the same selectivity observed in living cells in that the transferrin receptor traverses to the TGN at a much lower rate than mannose 6-phosphate receptors. Furthermore, recycling of mannose 6-phosphate/IGF-II receptors to the TGN requires GTP hydrolysis and can be distinguished biochemically from the constitutive transport of proteins between Golgi cisternae by its resistance to the weak base, primaquine.

  View details for Web of Science ID A1988Q697200013

  View details for PubMedID 2971452

• MANNOSE 6-PHOSPHATE RECEPTORS AND THEIR ROLE IN TARGETING PROTEINS TO LYSOSOMES JOURNAL OF MEMBRANE BIOLOGY Pfeffer, S. R. 1988; 103 (1): 7-16

  View details for Web of Science ID A1988P143300002

  View details for PubMedID 2972840

• A SINGLE RECEPTOR BINDS BOTH INSULIN-LIKE GROWTH FACTOR-II AND MANNOSE-6-PHOSPHATE SCIENCE MacDonald, R. G., Pfeffer, S. R., Coussens, L., Tepper, M. A., BROCKLEBANK, C. M., MOLE, J. E., Anderson, J. K., Chen, E., Czech, M. P., Ullrich, A. 1988; 239 (4844): 1134-1137

  Abstract

  Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate receptor (Man-6-P receptorCI), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for 125I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P-containing proteins.

  View details for Web of Science ID A1988M325400031

  View details for PubMedID 2964083

• Intracellular transport of the mannose-6-phosphate receptor. Progress in clinical and biological research Pfeffer, S. R., Goda, Y., CHEGE, N. W. 1988; 270: 365-375

  View details for PubMedID 2970646

• THE ENDOSOMAL CONCENTRATION OF A MANNOSE 6-PHOSPHATE RECEPTOR IS UNCHANGED IN THE ABSENCE OF LIGAND SYNTHESIS JOURNAL OF CELL BIOLOGY PFEFFER 1987; 105 (1): 229-234

  Abstract

  The cation-independent mannose-6-phosphate (Man-6-P) receptor is involved in the targeting of newly synthesized lysosomal hydrolases. To investigate the intracellular distribution of this receptor, a conjugate of lactoperoxidase coupled to asialoorosomucoid was used to catalyze its iodination within the endosomes of human hepatoma (HepG2) cells. The 215-kD, cation-independent Man-6-P receptor was iodinated by this procedure as shown by pentamannosyl-6-phosphate-Sepharose affinity chromatography and by immunoprecipitation of labeled cell extracts. The amount of this receptor detected in endosomes was found to be unchanged after inhibition of protein synthesis with cycloheximide. If the Man-6-P receptor accumulates in the Golgi apparatus in the absence of lysosomal hydrolase synthesis, it should have been correspondingly depleted from endosomes after a period of cycloheximide treatment, because these pools of receptor are in rapid equilibrium. Therefore, these data suggest that newly synthesized ligands are not required for the transport of the cation-independent Man-6-P receptor from the Golgi apparatus to endosomes.

  View details for DOI 10.1083/jcb.105.1.229

  View details for Web of Science ID A1987J342300024

  View details for PubMedID 3038925

  View details for PubMedCentralID PMC2114928

• BIOSYNTHETIC PROTEIN-TRANSPORT AND SORTING BY THE ENDOPLASMIC-RETICULUM AND GOLGI ANNUAL REVIEW OF BIOCHEMISTRY Pfeffer, S. R., Rothman, J. E. 1987; 56: 829-852

  View details for Web of Science ID A1987H963200026

  View details for PubMedID 3304148

• COMPONENTS RESPONSIBLE FOR TRANSPORT BETWEEN SUCCESSIVE GOLGI CISTERNAE ARE HIGHLY CONSERVED IN EVOLUTION JOURNAL OF BIOLOGICAL CHEMISTRY PAQUET, M. R., PFEFFER, BURCZAK, J. D., GLICK, B. S., ROTHMAN, J. E. 1986; 261 (10): 4367-4370

  Abstract

  Transport of a glycoprotein between compartments of the Golgi has been reconstituted in an in vitro system (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984) Cell 39, 405-416). Cytosolic components and ATP are absolutely required for transport. Here, we have tested the acceptor activity of Golgi fractions and of cytosolic fractions prepared from a variety of organisms. All mammalian Golgi fractions can act as "acceptor" in the in vitro assay. Similarly, the cytosol fractions obtained from plants as well as animals and a lower eukaryote substitute for the homologous CHO cytosol normally used. Moreover, a cytosol subfraction prepared from wheat germ complements a different cytosolic fraction obtained from bovine brain. Apparently, the essential components involved in the post-translational protein transport are remarkably conserved between plants, animals, and lower eukaryotes.

  View details for Web of Science ID A1986A778300003

  View details for PubMedID 3007452

• YEAST AND MAMMALS UTILIZE SIMILAR CYTOSOLIC COMPONENTS TO DRIVE PROTEIN-TRANSPORT THROUGH THE GOLGI-COMPLEX PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA DUNPHY, W. G., PFEFFER, CLARY, D. O., WATTENBERG, B. W., GLICK, B. S., ROTHMAN, J. E. 1986; 83 (6): 1622-1626

  Abstract

  Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

  View details for DOI 10.1073/pnas.83.6.1622

  View details for Web of Science ID A1986A582100018

  View details for PubMedID 3513182

  View details for PubMedCentralID PMC323135

• EPIDERMAL GROWTH-FACTOR - IS THE PRECURSOR A RECEPTOR NATURE PFEFFER, S., ULLRICH, A. 1985; 313 (5999): 184

  View details for DOI 10.1038/313184a0

  View details for Web of Science ID A1985AAB1100032

  View details for PubMedID 3871505

• RECONSTITUTION OF VESICLE-MEDIATED PROTEIN-TRANSPORT IN A CELL-FREE SYSTEM ROTHMAN, J., BRANDS, R., BURCZAK, J., DUNPHY, W., GLICK, B., PAQUET, M., PFEFFER, S., WATTENBERG, B. FEDERATION AMER SOC EXP BIOL. 1985: R9

  View details for Web of Science ID A1985ADF6100016

• THE SUBPOPULATION OF BRAIN COATED VESICLES THAT CARRIES SYNAPTIC VESICLE PROTEINS CONTAINS 2 UNIQUE POLYPEPTIDES CELL PFEFFER, KELLY, R. B. 1985; 40 (4): 949-957

  Abstract

  Coated vesicles have been purified in the past on the basis of their remarkably homogeneous structure, not their function. We have succeeded in isolating two subpopulations of bovine brain coated vesicles that carry specific "cargoes," in this case two synaptic vesicle membrane polypeptides (Mr = 95,000 and 65,000). Monoclonal antibodies that recognize cytoplasmic domains of these polypeptides can penetrate the clathrin coat and recognize them on the outer surface of the coated vesicle membrane. An immunoadsorption technique could therefore be used to fractionate coated vesicles on the basis of their membrane composition. The subpopulations have the normal complement of conventional coated vesicle proteins. Exclusive, however, to the subpopulations that carry synaptic vesicle polypeptides are two new coated vesicle polypeptides (Mr = 38,000 and 29,000).

  View details for DOI 10.1016/0092-8674(85)90355-1

  View details for Web of Science ID A1985AGH5400026

  View details for PubMedID 2859122