Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion

Abstract

The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for co-translational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains. A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides. Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes. However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel.

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

“…5), the directly adjacent OST and TRAP complexes spatially confine the C-terminal segment of Sec61a. This is in agreement with a rigid positioning of the C-terminal half of Sec61 on the ribosome in different functional states 15 . The N-terminal segment of Sec61a, in contrast, is spatially less constrained, which may be required to adopt the observed more open states during peptide translocation or membrane insertion 15 .…”

“…This is in agreement with a rigid positioning of the C-terminal half of Sec61 on the ribosome in different functional states 15 . The N-terminal segment of Sec61a, in contrast, is spatially less constrained, which may be required to adopt the observed more open states during peptide translocation or membrane insertion 15 . Notably, the Sec61 lateral gate, through which transmembrane helices of nascent membrane proteins are released into the membrane bilayer, points away from OST (Fig.…”

“…To analyse this interaction in more detail, we compared the subtomogram average to a reconstruction by cryo-EM SPA of wheatgerm RNCs bound to the mammalian translocon. Although detergent solubilization yielded primarily RNC-Sec61 complexes 15 , particle sorting also revealed an additional less-populated class that is similar to the subtomogram average (Fig. 4a).…”

“…To locate the Sec61 protein-conducting channel in the subtomogram average, a single-particle reconstruction of the canine ribosome (EMD 1480) 14 and an aligned atomic model of the nascent-chain-translocating canine Sec61 complex 15 were fitted into the subtomogram average as one rigid body. Near the ribosomal tunnel exit, a high-density region of the subtomogram average ( Fig.…”

“…13) allowed us to accurately position the protein-conducting channel in the translocon and to fit an atomic model of the nascent-chain-translocating heterotrimeric Sec61 complex ( Supplementary Fig. 13) 15 . Notably, the position and overall arrangement of the Sec61 complex in the presence of TRAP and OST remain indistinguishable from the canonical position observed previously for the ribosome-bound Sec61 or SecYEG complexes alone 17,18 .…”