Interaction mapping of the Sec61 translocon identifies two Sec61α regions interacting with hydrophobic segments in translocating chains

Abstract

Many proteins in organelles of the secretory pathway, as well as secretory proteins, are translocated across and inserted into the endoplasmic reticulum membrane by the Sec61 translocon, a protein-conducting channel. The channel consists of 10 transmembrane (TM) segments of the Sec61α subunit and possesses an opening between TM2b and TM7, termed the lateral gate. Structural and biochemical analyses of complexes of Sec61 and its ortholog SecY have revealed that the lateral gate is the exit for signal sequences and TM segments of translocating polypeptides to the lipid bilayer and also involved in the recognition of such hydrophobic sequences. Moreover, even marginally hydrophobic (mH) segments insufficient for membrane integration can be transiently stalled in surrounding Sec61α regions and cross-linked to them, but how the Sec61 translocon accommodates these mH segments remains unclear. Here, we used Cys-scanned variants of human Sec61α expressed in cultured 293-H cells to examine which channel regions associate with mH segments. A TM segment in a ribosome-associated polypeptide was mainly cross-linked to positions at the lateral gate, whereas an mH segment in a nascent chain was cross-linked to the Sec61α pore-interior positions at TM5 and TM10, as well as the lateral gate. Of note, cross-linking at position 180 in TM5 of Sec61α was reduced by an I179A substitution. We therefore conclude that at least two Sec61α regions, the lateral gate and the pore-interior site around TM5, interact with mH segments and are involved in accommodating them.

Signal sequences encode information for protein folding in the endoplasmic reticulum

“…By contrast, TMDs with insufficient hydrophobicity are transiently clogged at the Sec61 translocon and are eventually released into the ER lumen. This is consistent with the previous observation that insufficient hydrophobic sequences are stalled in the interior pore of the Sec61 translocon channel (Kida and Sakaguchi, 2018).…”

“…We began our studies by investigating how the Sec61 translocon deals with marginally hydrophobic sequences in proteins during their translocation into the ER. Earlier studies have shown that marginally hydrophobic sequences are neither hydrophobic enough to be inserted into the lipid bilayer nor hydrophilic enough to be translocated into the ER lumen, thus transiently stalling within the Sec61 translocon channel (Kida et al, 2016;Kida and Sakaguchi, 2018). To investigate the mechanism by which the translocon stalled nascent chain is cleared, we used our model C-terminally Venus-tagged substrates derived from WRB, which is a subunit of the tail-anchored membrane protein insertase ( Figure S1A) (Yamamoto and Sakisaka, 2012).…”

“…It is estimated that ~25% of TMDs of polytopic membrane proteins are marginally hydrophobic, and they are not suitable for insertion into the membrane by themselves in isolation (Elofsson and von Heijne, 2007;White and von Heijne, 2008). Recent studies have shown that weak TMDs can stall in the interior pore of the Sec61 translocon (Kida et al, 2016;Kida and Sakaguchi, 2018). In some cases, weak TMDs can be completely translocated into the ER lumen.…”

“…It is estimated that ~25% of TMDs of polytopic membrane proteins are marginally hydrophobic, meaning they are not suitable for insertion into the membrane by themselves in isolation (Elofsson and von Heijne, 2007; White and von Heijne, 2008). Recent studies have shown that marginally hydrophobic can transiently stall in the interior pore of the Sec61 translocon (Kida et al, 2016; Kida and Sakaguchi, 2018). It is unclear how and which factors assist in clearing the marginally hydrophobic TMD that is transiently retained at the Sec61 transocon during the co-translational membrane protein insertion.…”

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

“…Yet, some reports show the impact of cleavable SPs, or fragments thereof that are produced by the signal peptide peptidase that influence the folding of the downstream protein domain via specific chaperone recruitment or are used otherwise for the presentation of self-antigens via MHC class I [ 56 , 303 , 304 , 305 , 306 , 307 , 308 ]. Conclusions from research strategies such as in vitro protein import, photo-crosslinking studies, single Sec61 channel recordings from planar lipid bilayers experiments, and structural analyses of substrate-engaged Sec61 complexes have demonstrated the opening of the channel by SPs or TMHs of substrates, whereas SPs could be delivered co- or post-translationally [ 80 , 264 , 296 , 309 , 310 , 311 , 312 , 313 , 314 , 315 , 316 , 317 , 318 ]. To a certain degree, the process of opening the Sec61 complex by SPs and certain TMHs (excl.…”