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.

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Background:The molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) modulates gating of the polypeptide-conducting and calcium-permeable channel (Sec61 complex) in the membrane of the endoplasmic reticulum (ER). Results: Two co-chaperones, ERj3 and ERj6, support BiP in preventing ER calcium leakage via Sec61 complex. Conclusion: ERj3 and ERj6 facilitate Sec61 channel closing. Significance: Different co-chaperones assist BiP in Sec61 channel gating.

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon ‘breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.

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