Article

Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

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: 9.69). 07/2008; 181(7):1095-105. DOI: 10.1083/jcb.200804053
Source: PubMed

ABSTRACT 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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    • "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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    • "Proteinase K and Endo H protection of microsomal proteins Yeast microsomal membranes were prepared essentially as described previously (Kreft et al., 2006; Scott and Schekman, 2008). 15 OD 600 units of cells were harvested by centrifugation, resuspended in 1 ml resuspension buffer (10 mM Tris, pH 9.4, and 10 mM DTT), and incubated at room temperature for 10 min. "
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    ABSTRACT: Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.
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    • "Key events during ERAD include: substrate selection, retro-translocation and proteolysis of the misfolded proteins. Selection of ERAD substrates is typically carried out by molecular chaperones, and retro-translocation is thought to occur via one or more specialised retro-translocation channels [4] [5] [6]. "
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    ABSTRACT: The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.
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