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All Publications


  • A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex. Nature communications Hsieh, H., Lee, J. H., Chandrasekar, S., Shan, S. 2020; 11 (1): 5840

    Abstract

    Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.

    View details for DOI 10.1038/s41467-020-19548-5

    View details for PubMedID 33203865

  • A molecular recognition feature mediates ribosome-induced SRP-receptor assembly during protein targeting JOURNAL OF CELL BIOLOGY Fu, Y., Chandrasekar, S., Lee, J., Shan, S. 2019; 218 (10): 3307–19

    Abstract

    Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.

    View details for DOI 10.1083/jcb.201901001

    View details for Web of Science ID 000489119800014

    View details for PubMedID 31537711

    View details for PubMedCentralID PMC6781444

  • Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, J., Chandrasekar, S., Chung, S., Fu, Y., Liu, D., Weiss, S., Shan, S. 2018; 115 (24): E5487–E5496

    Abstract

    Signal recognition particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of ∼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.

    View details for DOI 10.1073/pnas.1802252115

    View details for Web of Science ID 000434933400009

    View details for PubMedID 29848629

    View details for PubMedCentralID PMC6004459

  • Structure of a prehandover mammalian ribosomal SRP.SRP receptor targeting complex SCIENCE Kobayashi, K., Jomaa, A., Lee, J., Chandrasekar, S., Boehringer, D., Shan, S., Ban, N. 2018; 360 (6386): 323–26

    Abstract

    Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-electron microscopy structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the guanosine triphosphate hydrolysis of SRP·SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon.

    View details for DOI 10.1126/science.aar7924

    View details for Web of Science ID 000430396600046

    View details for PubMedID 29567807

    View details for PubMedCentralID PMC6309883

  • Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ariosa, A., Lee, J., Wang, S., Saraogi, I., Shan, S. 2015; 112 (25): E3169–E3178

    Abstract

    The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.

    View details for DOI 10.1073/pnas.1422594112

    View details for Web of Science ID 000356731300008

    View details for PubMedID 26056263

    View details for PubMedCentralID PMC4485088