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Richard Zimmermann - One of the best experts on this subject based on the ideXlab platform.
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proteomics identifies signal peptide features determining the substrate specificity in human sec62 sec63 dependent er protein import
bioRxiv, 2019Co-Authors: Richard Zimmermann, Stefan Schorr, Duy Nguyen, Adolfo Cavalie, Markus Greiner, Petra Weissgerber, Sven Lang, Sarah Hassdenteufel, Johanna Dudek, Volkhard HelmsAbstract:Abstract In mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease. Databases The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).
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The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating.
Biology open, 2019Co-Authors: Anke Ziska, Richard Zimmermann, Adrienne W Paton, James C Paton, Johanna Dudek, Jörg Tatzelt, Sarah HassdenteufelAbstract:ABSTRACT The Sec61-complex as a dynamic polypeptide-conducting channel mediates protein transport into the human endoplasmic reticulum (ER) with the help of additional components. ER membrane resident Hsp40-type co-chaperone Sec63 as well as the ER lumenal Hsp70-type chaperone BiP were proposed to facilitate channel opening in a precursor-specific fashion. Here, we report on their rules of engagement in ER import of the prion protein (PrP) by addressing sixteen PrP-related variants which differ in their signal peptides and mature parts, respectively. Transport into the ER of semi-permeabilized human cells was analyzed upon depletion of the components by siRNA- or toxin-treatment. The results are consistent with the view of separate functions of BiP and Sec63 and strongly suggest that the co-chaperone/chaperone-pair facilitates Sec61 channel gating to the open state when precursor polypeptides with weak signal peptides in combination with detrimental features in the adjacent mature part were targeted. Thus, we expand the view of chaperone-mediated Sec61 channel gating by providing a novel example of a polybasic motif that interferes with signal peptide-mediated Sec61 channel gating. This article has an associated First Person interview with the first author of the paper.
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proteomics reveals signal peptide features determining the client specificity in human trap dependent er protein import
Nature Communications, 2018Co-Authors: Duy Nguyen, Volkhard Helms, Stefan Schorr, Sven Lang, Johanna Dudek, Regine Stutz, Stefan Pfeffer, Hudson H Freeze, Friedrich Forster, Richard ZimmermannAbstract:In mammalian cells, one-third of all polypeptides are transported into or across the ER membrane via the Sec61 channel. While the Sec61 complex facilitates translocation of all polypeptides with amino-terminal signal peptides (SP) or transmembrane helices, the Sec61-auxiliary translocon-associated protein (TRAP) complex supports translocation of only a subset of precursors. To characterize determinants of TRAP substrate specificity, we here systematically identify TRAP-dependent precursors by analyzing cellular protein abundance changes upon TRAP depletion using quantitative label-free proteomics. The results are validated in independent experiments by western blotting, quantitative RT-PCR, and complementation analysis. The SPs of TRAP clients exhibit above-average glycine-plus-proline content and below-average hydrophobicity as distinguishing features. Thus, TRAP may act as SP receptor on the ER membrane’s cytosolic face, recognizing precursor polypeptides with SPs of high glycine-plus-proline content and/or low hydrophobicity, and triggering substrate-specific opening of the Sec61 channel through interactions with the ER-lumenal hinge of Sec61α.
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Chaperone-Mediated Sec61 Channel Gating during ER Import of Small Precursor Proteins Overcomes Sec61 Inhibitor-Reinforced Energy Barrier
Cell reports, 2018Co-Authors: Sarah Hassdenteufel, Stephen High, Adrienne W Paton, James C Paton, Nicholas Johnson, Richard ZimmermannAbstract:Summary Protein transport into the mammalian endoplasmic reticulum (ER) is mediated by the heterotrimeric Sec61 channel. The signal recognition particle (SRP) and TRC systems and Sec62 have all been characterized as membrane-targeting components for small presecretory proteins, whereas Sec63 and the lumenal chaperone BiP act as auxiliary translocation components. Here, we report the transport requirements of two natural, small presecretory proteins and engineered variants using semipermeabilized human cells after the depletion of specific ER components. Our results suggest that hSnd2, Sec62, and SRP and TRC receptor each provide alternative targeting pathways for short secretory proteins and define rules of engagement for the actions of Sec63 and BiP during their membrane translocation. We find that the Sec62/Sec63 complex plus BiP can facilitate Sec61 channel opening, thereby allowing precursors that have weak signal peptides or other inhibitory features to translocate. A Sec61 inhibitor can mimic the effect of BiP depletion on Sec61 gating, suggesting that they both act at the same essential membrane translocation step.
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An Update on Sec61 Channel Functions, Mechanisms, and Related Diseases.
Frontiers in physiology, 2017Co-Authors: Sven Lang, Volkhard Helms, Adolfo Cavalie, Stefan Pfeffer, Friedrich Forster, Po-hsien Lee, Richard ZimmermannAbstract:The membrane of the endoplasmic reticulum (ER) of nucleated human cells harbors the protein translocon, which facilitates membrane integration or translocation of almost every newly synthesized polypeptide targeted to organelles of the endo- and exocytotic pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins and complexes that are permanently or transiently associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, modification of precursor polypeptides in transit through the Sec61 complex, and Sec61 channel gating, i.e., dynamic regulation of the pore forming subunit to mediate precursor transport and calcium efflux. Recently, cryoelectron tomography of translocons in native ER membrane vesicles, derived from human cell lines or patient fibroblasts, and even intact cells has given unprecedented insights into the architecture and dynamics of the native translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion, and translocation of newly synthesized polypeptides as well as the putative physiological roles of the Sec61 channel as a passive ER calcium leak channel. Furthermore, the structural insights into the Sec61 channel are incorporated into an overview and update on Sec61 channel-related diseases-the Sec61 channelopathies-and novel therapeutic concepts for their treatment.
Marc Nazaré - One of the best experts on this subject based on the ideXlab platform.
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Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway
PloS one, 2018Co-Authors: Wolfgang Klein, Claudia Rutz, Jamina Eckhard, Becky Provinciael, Edgar Specker, Martin Neuenschwander, Gunnar Kleinau, Patrick Scheerer, Jens-peter Von Kries, Marc NazaréAbstract:The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.
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use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic srp Sec61 targeting translocation pathway
PLOS ONE, 2018Co-Authors: Wolfgang Klein, Claudia Rutz, Jamina Eckhard, Becky Provinciael, Edgar Specker, Martin Neuenschwander, Gunnar Kleinau, Patrick Scheerer, Jens-peter Von Kries, Marc NazaréAbstract:The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.
Randy Schekman - One of the best experts on this subject based on the ideXlab platform.
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sec31 encodes an essential component of the copii coat required for transport vesicle budding from the endoplasmic reticulum
Molecular Biology of the Cell, 1997Co-Authors: Nina R Salama, John S Chuang, Randy SchekmanAbstract:The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae. This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150. Here, we report the cloning and characterization of the p150 gene. p150 is encoded by an essential gene. Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene. Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1. p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor. The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus. Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay.
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SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus.
The Journal of biological chemistry, 1994Co-Authors: Yann Esnault, David A Feldheim, Randy Schekman, Marc Blondel, François KépèsAbstract:Abstract The yeast SSS1 gene has been isolated as an extragenic high copy suppressor of Sec61, a mutant displaying defects in protein translocation into the endoplasmic reticulum (ER). We found that SSS1 is an essential gene required for transfer of secretory precursors through the ER membrane. Here we demonstrate that the SSS1 product (Sss1p) is firmly bound to the ER membrane and exposes its amino-terminal half on the cytosolic side. Only detergent, or an alkali treatment, is effective at extracting Sss1p from the membrane. Coimmunoprecipitation experiments revealed that Sss1p and Sec61p participate in the same multisubunit complex. Cross-linking followed by immunoprecipitation specifically yielded an additional polypeptide of molecular mass 73 kDa. Moreover, Sss1p and Sec61p show mutually stabilizing interactions: Sss1p is destabilized in a Sec61 mutant context, and mutated Sec61p is stabilized by Sss1p overproduction. These observations account for the isolation of SSS1 as a dosage-dependent suppressor of Sec61. Since the polytopic integral membrane protein Sec61p is adjacent to translocating precursors and to ribosomes, and given the comparable translocation deficiencies of sss1 or Sec61 mutants, we propose that Sss1p belongs to the "Sec61 subcomplex" that constitutes the pore of the membrane-bound translocation apparatus.
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Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.
Molecular and Cellular Biology, 1992Co-Authors: David A Feldheim, Jonathan Rothblatt, Randy SchekmanAbstract:SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.
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Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.
Molecular and Cellular Biology, 1992Co-Authors: David A Feldheim, Jonathan Rothblatt, Randy SchekmanAbstract:SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.
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protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum
Molecular Biology of the Cell, 1992Co-Authors: C Stirling, Jonathan Rothblatt, Midori Hosobuchi, Raymond J Deshaies, Randy SchekmanAbstract:Abstract Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (Sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of Sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The Sec61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.
William R. Skach - One of the best experts on this subject based on the ideXlab platform.
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cotranslational stabilization of sec62 63 within the er Sec61 translocon is controlled by distinct substrate driven translocation events
Molecular Cell, 2015Co-Authors: Brian J. Conti, Prasanna K. Devaraneni, Zhongying Yang, Larry L. David, William R. SkachAbstract:The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αβγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62, and Sec63. Moreover, Sec62/63 stabilization within the translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. These data show that cotranslational translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.
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Cotranslational Stabilization of Sec62/63 within the ER Sec61 Translocon Is Controlled by Distinct Substrate-Driven Translocation Events
Molecular cell, 2015Co-Authors: Brian J. Conti, Prasanna K. Devaraneni, Zhongying Yang, Larry L. David, William R. SkachAbstract:The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αβγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62, and Sec63. Moreover, Sec62/63 stabilization within the translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. These data show that cotranslational translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.
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stepwise insertion and inversion of a type ii signal anchor sequence in the ribosome Sec61 translocon complex
Cell, 2011Co-Authors: Prasanna K. Devaraneni, Brian J. Conti, Zhongying Yang, Yoshihiro Matsumura, Arthur E Johnson, William R. SkachAbstract:In eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility, and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. Importantly, type II SA inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment.
Karin Römisch - One of the best experts on this subject based on the ideXlab platform.
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How does Sec63 affect the conformation of Sec61 in yeast?
PLoS computational biology, 2021Co-Authors: Pratiti Bhadra, Lalitha Yadhanapudi, Karin Römisch, Volkhard HelmsAbstract:The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the accessory membrane protein Sec62 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.
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Model: Role of the Sec61 N-terminal helix in posttranslational ER import.
2019Co-Authors: Francesco Elia, Lalitha Yadhanapudi, Thomas Tretter, Karin RömischAbstract:Left: TMD1 of Sec61 (red) is anchored at its N-terminus in the ER membrane by the strongly hydrophobic, amphipathic N-terminal helix H1 which positions Sec61TMD1 such that it can interact closely with TMD3 of Sec63 (blue) in wildtype S. cerevisiae. Middle: Deletion of the N-terminal acetylation site (yellow) and H1 of Sec61 in Sec61ΔN21p reduces Sec61TMD1 anchoring to the membrane and thereby changes its tilt angle and/or mobility, thus reducing the interaction between Sec61TMD1 with Sec63TMD3. Right: Deletion of H1 in Sec61ΔH1p additionally increases the distance between Sec61TMD1 and Sec63TMD3 because of the repositioning of the acetyl group (which serves as an anchor for a putative ERAD factor) (green) close to the Sec63-binding residues of Sec61TMD1, thereby blocking Sec61p-Sec63p interaction. Both middle and right scenarios affect the opening of the Sec61 lateral gate by Sec63p and hence posttranslational import.
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Optimization of crosslinking to Sec61S353C.
2019Co-Authors: Fabio Pereira, Mandy Rettel, Frank Stein, Mikhail M. Savitski, Ian Collinson, Karin RömischAbstract:A: Topological model of Sec61. B: Comparison of crosslinking patterns to Sec61 versus Sec61S353C with cysteine- and NH2-reactive SMPH. 17 eq microsomes per lane were crosslinked with 1 mM SMPH on ice and proteins resolved by SDS-PAGE. Sec61 was detected with an antibody against its N-terminus. Note that both Sec61 and Sec61S353C crosslink to Sss1. Additional crosslinked bands occurring in Sec61S353C samples are indicated by arrows in Sec61 panel. The largest product consists of Sec61S353C crosslinked to Sec63 (right panel). C: Sec61S353C crosslinking with SMPH (non-cleavable) or LC-SPDP (cleavable). Crosslinking was done as above and samples were resolved on SDS-PAGE without or with 200 mM DTT in the sample buffer as indicated. D: Crosslinking to Sec61S353C after microsome extraction. Microsomes (17 eq/lane) were extracted as indicated or mock-treated, crosslinked as above, and Sec61S353C and crosslinking products detected with an antibody against the Sec61 N-terminus. Note that crosslinks to Sss1 and Sec63 are sensitive to carbonate-extraction. E: Crosslinking of His14-Sec61S353C microsomes with LC-SPDP. Crosslinking was done as above. Note that the N-terminal His14-tag did not affect crosslinking to Sec63 or Sss1 indicating no gross conformational alterations in the Sec61 complex. The asterisk indicates a non-specific band occuring independently of crosslinking in the Sec61 blot.
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The N-terminal helix of Sec61p is required for Sec61 complex stability.
2019Co-Authors: Francesco Elia, Lalitha Yadhanapudi, Thomas Tretter, Karin RömischAbstract:A: Stability of heptameric Sec complexes in Sec61ΔH1 and Sec61ΔN21 membranes. Wildtype, Sec61ΔH1, and Sec61ΔN21 microsomes were solubilized in solubilization buffer (see Methods) containing 3% digitonin and lysates centrifuged at 110,000 g to sediment ribosome-bound Sec61 complexes (ribosome-associated membrane proteins, RAMP). From the supernatants, heptameric Sec complexes were precipitated with Concanavalin A Sepharose (Con A). Sec61p and Sec63p not bound to either Con A or ribosomes were detected in the “Free” fraction. In all fractions, Sec61p and Sec63p were detected by immunoblotting with polyclonal antisera. Loss of Sec61p from the Sec61ΔH1 and Sec61ΔN21 Soluble fractions is likely due to the formation of SDS-resistant aggregates in digitonin. B: Stability of Sec61 complexes in Sec61ΔH1 and Sec61ΔN21 strains. Microsomes were solubilized in 1% Triton X-100 and layered onto a 0–15% sucrose gradient. After centrifugation at 200,000 g for 16 h, fractions were collected from top to bottom, and proteins resolved by SDS-PAGE. Sec61p, Sbh1p, and Sss1p were detected by immunoblotting. For both A and B the experiment was performed 4 times.
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Effect of Sec61 interaction with Mpd1 on endoplasmic reticulum-associated degradation
2019Co-Authors: Fabio Pereira, Mandy Rettel, Frank Stein, Mikhail M. Savitski, Ian Collinson, Karin RömischAbstract:Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we aimed to identify new lumenal interaction partners of the Sec61 channel by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.
