Abstract
In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment.
Highlights
In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved proteinconducting channel Sec[61], which has been structurally studied in isolated, detergentsolubilized states
For structure determination of the ribosome-bound translocon in its native membrane environment, we applied Cryo-electron tomography (CET) and subtomogram analysis to rough ER (rER) vesicles isolated from canine pancreas
Functional assays and western blotting have shown that these rER vesicles contain all protein components necessary for co-translational protein transport and membrane protein integration, as well as subsequent maturation steps of nascent peptides[11,12]
Summary
Secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved proteinconducting channel Sec[61], which has been structurally studied in isolated, detergentsolubilized states. Recent revolutionary developments in direct detector technology[16] suggest that subnanometer resolution subtomogram averages are in reach for much smaller data sets and for non-symmetrical macromolecules Benefitting from these developments in direct detector technology, we obtained a subtomogram average of mainly idle mammalian ribosomes bound to the native translocon in rER vesicles at subnanometer resolution. This structure allows determining the conformation of native Sec[61] in a non-inserting state, revealing an open lateral gate even in the absence of a nascent transmembrane helix, breaking with the current dogma of ER translocation
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