Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Abstract

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.The Sec61/SecY translocon complex mediates translocation of proteins across and integration into the endoplasmic reticulum (ER) 3 membrane (1, 2). Proteins are targeted to this complex by hydrophobic signal sequences (3), and the signals and subsequent hydrophobic segments of the polypeptide are integrated into the bilayer as transmembrane helices. The translocon consists of Sec61␣ (Sec61p in yeast) with 10 transmembrane domains, and the single spanning proteins Sec61␤ (Sbh1p) and Sec61␥ (Sss1p), corresponding in bacteria to SecY/ SecG/SecE. The crystal structure of the SecY complex of the archaebacterium Methanococcus jannaschii (4) revealed a compact helix bundle that can form a hydrophilic channel with a lateral exit site. The central passage is blocked by a lumenal plug domain and a central constriction ring. To open, the plug needs to move out, and the ring must expand (5, 6). The crystal structure and cross-linking experiments (7) suggest that the translocation channel is formed by a single heterotrimer, although it has been proposed that upon opening of the channel two or more complexes may cooperate (8, 9).The simplest case of protein topogenesis is the orientation of a signal sequence upon entering the translocon. Cleavable signal sequences assume a N cyt /C exo orientation (cytoplasmic N and exoplasmic C terminus), whereas noncleavable signal-anchors of membrane proteins insert in N exo /C cyt or N cyt /C exo direction to translocate either their N-or their C-terminal end, respectively. Best established is the role of charged residues on either side of the hydrophobic core of the signal, which according to the "positive-inside rule" (10, 11) generally results in the more positive end to be cytosolic. Additional determinants are the folding of sequences N-terminal to an internal signal that may sterically hinder N-translocation (12) and the ...

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

“…A variety of different mutations have previously been found to mediate the suppression of signal peptide defects, the prl ('protein localization') phenotype described in bacteria (Emr et al, 1981;Junne et al, 2007;Smith et al, 2005) and yeast (Junne et al, 2007). prl mutations specifically destabilize the closed state of the translocon, facilitating signal entry and initiation of peptide insertion and translocation.…”

“…Sequences were assembled using the Newbler 2.6 assembler with default options, except for specifying the scaffolding and four processors. A total of 50 scaffolds were generated, with an average size of 843,301 bp and an N50 of 2,374,876 bp (the average contig size within these scaffolds was 124,629 bp; N50 scaffold contig size These Sec61 mutants have previously been characterized by Junne et al (Junne et al, 2006;Junne et al, 2007;Junne et al, 2010). prl phenotype and CPY translocation defects were analyzed by Junne et al (Junne et al, 2007).…”

“…This indicates similar, yet distinct, modes of action of the deca-and heptadepsipeptide inhibitors on Sec61. In addition, we also tested our existing library of Sec61p mutants that had been isolated in the context of membrane protein topogenesis (Junne et al, 2007) with respect to sensitivity to compounds 1 and 2 (supplementary material Fig. S3).…”

“…In prl mutants this state is destabilized, resulting in reduced binding affinity for the compounds. The Sec61p mutants isolated as resistant to compound 1 were tested for the prl phenotype and CPY translocation defects as in Junne et al (Junne et al, 2007). S, sensitive; R, resistant (IC 50 at least 50-fold higher than wild-type); (R), moderately resistant (IC 50 at least two-fold higher than wild-type).…”

Stitching proteins into membranes, not sew simple

“…The charge distribution of residues flanking SA sequences favours positively charged amino acids on the cytoplasmic side of the membrane (von Heijne and Gavel, 1988;Hartmann et al, 1989). They could possibly assert their topogenic effects by electrostatic interactions using negatively charged molecules (amino acids in the translocon or lipid head groups) on the cytoplasmic side of the ER membrane as 'anchorage' points (Junne et al, 2007;Bogdanov et al, 2008). However, in reality, lipids are likely to play a much more complex and dynamic role in exerting their topological effects [reviewed in Bogdanov et al (2014)].…”