Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane 1 , 2 . Proteins with hydrophobic signal sequences are first recognized by the signal recognition particle (SRP) 3 , 4 and then moved co-translationally through the Sec61/SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass SRP and are moved through the channel post-translationally 5 , 6 . In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62/Sec63 complex 7 – 9 , and substrates are moved through the channel by the luminal BiP ATPase 9 . How the Sec62/63 complex activates the Sec61 channel for post-translational translocation is unclear. Here, we report the electron cryo-microscopy (cryo-EM) structure of the Sec complex from S. cerevisiae , consisting of the Sec61 channel and the Sec62, Sec63, Sec71, and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel’s plug domain. Lateral channel opening is triggered by Sec63 interacting with both cytosolic loops in the C-terminal half of Sec61 and trans-membrane (TM) segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70 (ref. 10 ). Our structure shows how the Sec61 channel is activated for post-translational protein translocation.

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SUMMARY Lipid regulation of ion channels by low-abundance signaling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) has emerged as a central cellular mechanism for controlling ion channels and the excitability of nerves. A lack of robust assays suitable for facile detection of a lipid bound to a channel has hampered the probing of the lipid binding sites and measuring the pharmacology of putative lipid agonists for ion channels. Here, we show a fluorescent PIP2 competition assay for detergent-purified potassium channels, including TWIK-1-related K+-channel (TREK-1). Anionic lipids PA and phosphatidylglycerol (PG) bind dose dependently (9.1 and 96 mM, respectively) and agonize the channel. Our assay shows PIP2 binds with high affinity (0.87 mM) but surprisingly can directly antagonize TREK-1 in liposomes. We propose a model for TREK-1 lipid regulation where PIP2 can compete with PA and PG agonism based on the affinity of the lipid for a site within the channel.

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