Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum

Schorr, Stefan; Klein, Marie-Christine; Gamayun, Igor; Melnyk, Armin; Jung, Martin; Schäuble, Nico; Wang, Qian; Hemmis, Birgit; Bochen, Florian; Greiner, Markus; Lampel, Pavel; Urban, Sabine; Haßdenteufel, Sarah; Dudek, Johanna; Chen, Xing-Zhen; Wagner, Richard; Cavalié, Adolfo; Zimmermann, Richard

doi:10.1074/jbc.m115.636639

Cited by 33 publications

(49 citation statements)

References 73 publications

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“…Furthermore, we note that the full scans of the blots are shown at https://data.mendele y.com/datasets/6s5hn73jcv/2 and that the particular western blot was done only once. However, the high reproducibility of western blots for ERj3, Grp170, and BiP in human cells was previously demonstrated [23,53]. Sec63 in CRISPR/Cas9-treated HEK293 cells caused more effective accumulation of pre-ERj3 in the ER membrane and concomitant inhibition of ERj3 import into the ER (Fig.…”

Section: Depletion Of Bip Inhibits Import Of Pre-erj3 Into the Ermentioning

confidence: 75%

“…We note that the full scans of the blots are shown at https://data.mendeley.com/datasets/ 6s5hn73jcv/2 and that the particular western blot was done only once. However, the high reproducibility of western blots for ERj3, Grp170, Sec62, BiP, and calreticulin in human cells was previously demonstrated [18][19]23,53] amino acid residues at the amino termini of helices 2 ( 42 KKAYRK) and 4 ( 82 KRK) ( Fig. 10A).…”

Section: Depletion Of Bip Inhibits Import Of Pre-erj3 Into the Ermentioning

confidence: 80%

“…The antibody directed against ERj3 was affinity-purified on Sul-foLink-immobilized peptide (Thermo Fisher Scientific). Antibody quality was previously documented [53,54]. We note that the full scans of the blots are shown in Supplementary information (https://data.mendeley.com/datasets/ 6s5hn73jcv/2).…”

Section: Methodsmentioning

confidence: 99%

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Identification of signal peptide features for substrate specificity in human Sec62/Sec63‐dependent ER protein import

et al. 2020

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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 22 novel Sec62/Sec63 substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy-terminal region of SP (C-region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP 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 immunoglobulin heavy-chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61-channel inhibitor CA7M741. Thus, the human Sec62/Sec63 complex may support Sec61-channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino-terminal peptide of Sec61a or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61a. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked polycystic liver disease.

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Section: Depletion Of Bip Inhibits Import Of Pre-erj3 Into the Ermentioning

confidence: 75%

Section: Depletion Of Bip Inhibits Import Of Pre-erj3 Into the Ermentioning

confidence: 80%

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Identification of signal peptide features for substrate specificity in human Sec62/Sec63‐dependent ER protein import

et al. 2020

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“…We purchased murine monoclonal antibodies against β-actin (Sigma, A5441, used dilution 1:10,000), HA-tag (Biolegend, 901515, used dilution 1:1,000), the mycDDK-tag (Origene, TA50011), and rabbit polyclonal antibodies against FKBP10 (Proteintech, 12172, used affinity purified on Sulfolink-immobilized peptide (Thermo Fisher Scientific). Antibody quality was previously documented [53,54]. We note that the full scans of the blots are shown in Supplementary Information http://dx.doi.or/10.17632/6s5hn73jcv.1.…”

Section: Methodsmentioning

confidence: 99%

Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

Zimmermann

Lang

Schorr

et al. 2019

Preprint

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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.DatabasesThe 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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“…During protein translocation, the lateral gate is typically occupied by a signal peptide and the central aqueous pore by the polypeptide chain in transit. Notably, efficient closing of the Sec61-channel can also involve allosteric effectors, such as BiP with its ER-lumenal Hsp40-type cochaperones ERj3 plus ERj6 or Ca 2+ -bound Sec62 plus Ca 2+ -CaM[58,95,101,102].2513 FEBS Letters 593 (2019) 2506-2524 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.…”

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confidence: 99%

ER import of small human presecretory proteins: components and mechanisms

et al. 2019

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Protein transport into the mammalian endoplasmic reticulum (ER) used to be seen as strictly cotranslational, that is temporarily and mechanistically coupled to protein synthesis. In the course of the last decades, however, several classes of precursors of soluble and membrane proteins were found to be post‐translationally imported into the ER, without any involvement of the ribosome. The first such class to be identified were the small presecretory proteins; tail‐anchored membrane proteins followed next. In both classes, the inherent address tag is released from the translating ribosome before the initiation of ER import, as part of the fully synthesized precursor. In small presecretory proteins, the information for ER targeting and ‐translocation via the polypeptide‐conducting Sec61‐channel is encoded by a classical N‐terminal signal peptide, which is released from the ribsosome before targeting due to the small size of the full‐length precursor. Here, we discuss the current state of research on targeting and translocation of small presecretory proteins into the mammalian ER. In closing, we present a unifying hypothesis for ER protein translocation in terms of an energy diagram for Sec61‐channel gating.

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Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum

Cited by 33 publications

References 73 publications

Identification of signal peptide features for substrate specificity in human Sec62/Sec63‐dependent ER protein import

Identification of signal peptide features for substrate specificity in human Sec62/Sec63‐dependent ER protein import

Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

ER import of small human presecretory proteins: components and mechanisms

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