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 , University of California, Berkeley, Berkeley, California, United States
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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    ABSTRACT: Giardia lamblia is a flagellated protozoan enteroparasite transmitted as an environmentally resistant cyst. Trophozoites attach to the small intestine of vertebrate hosts and proliferate by binary fission. They access nutrients directly via uptake of bulk fluid phase material into specialized endocytic organelles termed peripheral vesicles (PVs), mainly on the exposed dorsal side. When trophozoites reach the G2/M restriction point in the cell cycle they can begin another round of cell division or encyst if they encounter specific environmental cues. They induce neogenesis of Golgi-like organelles, encystation-specific vesicles (ESVs), for regulated secretion of cyst wall material. PVs and ESVs are highly simplified and thus evolutionary diverged endocytic and exocytic organelle systems with key roles in proliferation and transmission to a new host, respectively. Both organelle systems physically and functionally intersect at the endoplasmic reticulum (ER) which has catabolic as well as anabolic functions. However, the unusually high degree of sequence divergence in Giardia rapidly exhausts phylogenomic strategies to identify and characterize the molecular underpinnings of these streamlined organelles. To define the first proteome of ESVs and PVs we used a novel strategy combining flow cytometry-based organelle sorting with in silico filtration of mass spectrometry data. From the limited size datasets we retrieved many hypothetical but also known organelle-specific factors. In contrast to PVs, ESVs appear to maintain a strong physical and functional link to the ER including recruitment of ribosomes to organelle membranes. Overall the data provide further evidence for the formation of a cyst extracellular matrix with minimal complexity. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000694.
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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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    ABSTRACT: Insulin is an essential hormone for maintaining metabolic homeostasis in the body. To make fully bioactive insulin, pancreatic beta cells initiate synthesis of the insulin precursor, preproinsulin, at the cytosolic side of the endoplasmic reticulum (ER), whereupon it undergoes co- and post-translational translocation across the ER membrane. Preproinsulin is cleaved by signal peptidase to form proinsulin that folds on the luminal side of the ER, forming three evolutionarily conserved disulfide bonds. Properly folded proinsulin forms dimers and exits from the ER, trafficking through Golgi complex into immature secretory granules wherein C-peptide is endoproteolytically excised, allowing fully bioactive two-chain insulin to ultimately be stored in mature granules for insulin secretion. Although insulin biosynthesis has been intensely studied in recent decades, the earliest events, including proinsulin entry and exit from the ER, have been relatively understudied. However, over the past 5 years, more than 20 new insulin gene mutations have been reported to cause a new syndrome termed Mutant INS-gene-induced Diabetes of Youth (MIDY). Although these mutants have not been completely characterized, most of them affect proinsulin entry and exit from the ER. Here, we summarize our current knowledge about the early events of insulin biosynthesis and review recent advances in understanding how defects in these events may lead to pancreatic beta cell failure.
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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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