Scott, D. C. & Schekman, R. Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins. J. Cell Biol. 181, 1095-1105

Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 07/2008; 181(7):1095-105. DOI: 10.1083/jcb.200804053
Source: PubMed


Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin-proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.

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    • "Directed translocation of proteins across ESV membranes towards the cytosol as part of a quality control system was inferred from the observation that proteasome complexes were recruited to the vicinity of developing ESV organelles [26]. Pore complexes such as Sec61, for which an alpha and a gamma subunit are annotated in the Giardia genome database, are required for co-translational insertion and are also strongly implicated in retro-translocation to the cytoplasm [65]. In support of co-translational insertion of proteins across ESV membranes, a signal recognition particle component, 54 kDa protein (Gl15156), has been localized partially to ESVs (Figure 8B). "
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    • "In ERAD, the misfolded protein is first recognized by binding to ER chaperones. The misfolded protein is then retrotranslocated from the ER to the cytosol (Fig. 2.5; Pilon, Schekman, & Romisch, 1997; Plemper, Bohmler, Bordallo, Sommer, & Wolf, 1997; Scott & Schekman, 2008). If disulfide bonds have been formed, it is believed that they must be broken before retrotranslocation , a process that is likely to involve the reductase function of some of the PDI family members (Grubb, Guo, Fisher, & Brodsky, 2012; Moore, Bernardi, & Tsai, 2010; Walczak, Bernardi, & Tsai, 2012). "
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    • "We next investigated the fate of Deg1:Sec62p, an ERAD substrate with two transmembrane domains and both termini in the cytoplasm, using cycloheximide chase experiments [32]. The cytosolic N-terminus of Deg1:Sec62p contains an N-glycosylation acceptor site which during ERAD is translocated into the ER lumen and modified [32]. Unfortunately, the protein was poorly expressed in our strain background so the determination of its exact half life was problematic, and although we repeated the experiment several times, expression could not be improved. "
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    ABSTRACT: The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role. We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export. A complete deletion of L7 in Sec61p resulted in viable, cold- and tunicamycin-hypersensitive yeast cells with strong defects in posttranslational protein import of soluble proteins into the ER, and in ERAD of soluble substrates. Membrane protein ERAD was only moderately slower in sec61[increment]L7 than in wildtype cells. Although Sec61[increment]L7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61[increment]L7 channels, and formation of hetero-heptameric Sec complexes were not affected. We conclude that L7 of Sec61p is required for initiation of posttranslational soluble protein import into and misfolded soluble protein export from the ER, suggesting a key role for L7 in transverse gating of the Sec61 channel.
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