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

Zimmermann, R.; Lang, S.; Schorr, S.; Hassdenteufel, S.; Cavalie, A.; Greiner, M.; Weissgerber, P.; Dudek, J.; Nguyen, D.; Helms, V.; Foerster, F.; Nagaraj, N.; Paton, J. C.; Paton, A. W.; Molinari, M.; Loi, M.

doi:10.1101/867762

bioRxiv · 10.1101/867762

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

Zimmermann, R.·Lang, S.·Schorr, S.·Hassdenteufel, S.·Cavalie, A.·Greiner, M.·Weissgerber, P.·Dudek, J.·Nguyen, D.·Helms, V.·Foerster, F.·Nagaraj, N.·Paton, J. C.·Paton, A. W.·Molinari, M.·Loi, M.

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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 Sec61 or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61. 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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Zimmermann, R., Lang, S., Schorr, S., Hassdenteufel, S., Cavalie, A., Greiner, M., Weissgerber, P., Dudek, J., Nguyen, D., Helms, V., Foerster, F., Nagaraj, N., Paton, J. C., Paton, A. W., Molinari, M., Loi, M.. 2019-12-06. Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import. https://doi.org/10.1101/867762

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