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SND3 is the membrane insertase within a distinct SEC61 translocon complex

ABSTRACT: During the biogenesis of most eukaryotic integral membrane proteins (IMPs), transmembrane domains are inserted into the endoplasmic reticulum membrane by a dedicated insertase or the SEC61 translocon. The SRP-independent (SND) pathway is the least understood route into the membrane, despite catering for a broad range of IMP types. Here, we show that Chaetomium thermophilum SND3 is a membrane insertase with an atypical fold. We further present a cryo-electron microscopy structure of a ribosome-associated SND3 translocon complex involved in co-translational IMP insertion. The structure reveals that the SND3 translocon additionally comprises the complete SEC61 translocon, CCDC47 and TRAPɑ. Here, the SEC61β N-terminus works together with CCDC47 to prevent substrate access to the translocon. Instead, molecular dynamics simulations show that SND3 disrupts the lipid bilayer to promote IMP insertion via its membrane-embedded hydrophilic groove. Structural and sequence comparisons indicate that the SND3 translocon is a distinct multipass translocon in fungi, euglenozoan parasites and other eukaryotic taxa.

INSTRUMENT(S):

ORGANISM(S): Chaetomium Thermophilum

SUBMITTER: Julian Langer

LAB HEAD: Julian Langer

PROVIDER: PXD060914 | Pride | 2025-09-24

REPOSITORIES: Pride

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			Action	DRS
	240207_2505_TzYang_25min_DDA_Sample-1_Slot2-37_1_733.d.zip	Other
	240207_2505_TzYang_25min_DDA_Sample-2_Slot2-38_1_734.d.zip	Other
	240207_2505_TzYang_25min_DDA_Sample-3_Slot2-39_1_735.d.zip	Other
	240207_2505_TzYang_25min_DDA_Sample-4_Slot2-40_1_736.d.zip	Other
	240207_2505_TzYang_25min_DDA_Sample-5_Slot2-41_1_737.d.zip	Other

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Project description:Maintenance of cellular homeostasis and functionality requires rapid translocation of newly synthesized lipids across the membrane leaflets of the endoplasmic reticulum (ER) where majority of cellular lipid biosynthesis takes place. This process is facilitated without the need for ATP by specific membrane proteins—scramblases—a few of which have been very recently identified in the ER. We have previously resolved the structure of the translocon-associated protein (TRAP) bound to the Sec61 translocon, and found this complex to render the membrane locally thinner. We thus hypothesized that the translocon could provide scrambling pathways in the ER membrane. Here, we first observed non-selective lipid scrambling by reconstituted translocon complexes using complementary fluorescence approaches. Our experiments with inhibitors that block the putative Sec61 scrambling site suggest that an additional scrambling pathway is active in the translocon. Our extensive molecular dynamics simulations indicate that this additional pathway could be provided by the trimeric bundle of TRAP subunits. Its crevice rich in polar residues shields lipid head groups as they traverse the membrane via a credit card mechanism. We analyzed the kinetics and thermodynamics of lipid scrambling by both Sec61 and TRAP and demonstrated that local membrane thinning provides a key contribution to scrambling efficiency. Both proteins appear selective towards phosphatidylcholine lipids over phosphatidylethanolamine and phosphatidylserine, though this trend reflects the natural flip-flop tendencies of these lipids in a protein-free membrane. The identified scrambling pathway in the Sec61 translocon is located at the lateral gate region, which is likely either closed or occupied by a nascent polypeptide during protein translocation. Moreover, our simulations suggest Sec61 scrambling to be impeded by physiological salt concentration. Together with the observations with Sec61 inhibitors, this indicates the presence of an alternative scrambling site in the translocon complex, and the metazoan-specific transmembrane helix bundle of TRAPβ, TRAPγ, and TRAPδ seems like a viable candidate with scrambling activity that is insensitive to the translocon functional state and solvent conditions.

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SND3 is the membrane insertase within a distinct SEC61 translocon complex

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