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.

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.

Summary Eeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca 2+ homeostasis by enhancing the Ca 2+ leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca 2+ leakage of ES1 analogs studied and induced Ca 2+ -dependent cell death. Using small interfering RNA-mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca 2+ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca 2+ -permeable, open state, in which Sec61 complexes allow Ca 2+ leakage but are translocation incompetent.

To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate. Heterologous expression of SLC35B1 in E. coli reveals that SLC35B1 is highly specific for ATP and ADP and acts in antiport mode. Moreover, depletion of SLC35B1 from HeLa cells reduces ER ATP levels and, as a consequence, BiP activity. Thus, human SLC35B1 may provide ATP to the ER and was named AXER (ATP/ADP exchanger in the ER membrane). Furthermore, we propose an ER to cytosol low energy response regulatory axis (termed lowER) that appears as central for maintaining ER ATP supply.

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.