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A Complex of Pdi1p and the Mannosidase Htm1p Initiates Clearance of Unfolded Glycoproteins from the Endoplasmic Reticulum

Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, 8093 Zurich, Switzerland.
Molecular cell (Impact Factor: 14.46). 06/2011; 42(6):782-93. DOI: 10.1016/j.molcel.2011.04.027
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ABSTRACT Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man(8)GlcNAc(2) carbohydrates bound to unfolded proteins, yielding Man(7)GlcNAc(2). This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathways and how the flux through these pathways is adjusted to stress conditions.

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    • "Genetic data imply that the Hrd3/Yos9 heterodimer is involved in this process (Denic et al., 2006; Gauss et al., 2006a; Izawa et al., 2012). The lectin Yos9 preferentially binds N-linked glycan structures on aberrant glycoproteins, which are most likely generated by the sequential action of the mannosidases Mns1 and Htm1 (Quan et al., 2008; Gauss et al., 2011). The activity of these enzymes is thought to destine unfolded proteins for ERAD and thereby delimitates the time a newly imported glycoprotein is given to attain its native conformation in the ER. "
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    ABSTRACT: Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase) and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR, which is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70 type chaperone Kar2 and the Hsp40 type cochaperone Scj1 bind to Hrd3KR. Noteworthy, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream acting components like Der1. © 2014 by The American Society for Cell Biology.
    Molecular Biology of the Cell 11/2014; 26(2). DOI:10.1091/mbc.E14-07-1202 · 4.55 Impact Factor
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    • "The lectins Htm1p and Yos9p are both essential for ERAD in yeast [8], [9], [10]. Htm1p trims substrate’s high mannose oligosaccharides to expose α1,6 mannose moieties [11], [12], [13], which can then be recognized through the mannose-6-phosphate receptor homology (MRH) domain of Yos9p [14], [15], [16]. Yos9p also interacts with Hrd3p, the interaction partner of the ubiquitin ligase Hrd1p [17], [18], thus permitting substrates to be delivered from Yos9p to Hrd1p via Hrd3p [19], [20]. "
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    ABSTRACT: Misfolded proteins of the endoplasmic reticulum (ER) are eliminated by the ER-associated degradation (ERAD) in eukaryotes. In S. cerevisiae, ER-resident lectins mediate substrate recognition through bipartite signals consisting of an unfolded local structure and the adjacent glycan. Trimming of the glycan is essential for the directional delivery of the substrates. Whether a similar recognition and delivery mechanism exists in mammalian cells is unknown. In this study, we systematically study the function and substrate specificity of known mammalian ER lectins, including EDEM1/2/3, OS-9 and XTP-3B using the recently identified ERAD substrate sonic hedgehog (SHH), a soluble protein carrying a single N-glycan, as well as its nonglycosylated mutant N278A. Efficient ERAD of N278A requires the core processing complex of HRD1, SEL1L and p97, similar to the glycosylated SHH. While EDEM2 was required for ERAD of both glycosylated and non-glycosylated SHHs, EDEM3 was only necessary for glycosylated SHH and EDEM1 was dispensable for both. Degradation of SHH and N278A also required OS-9, but not the related lectin XTP3-B. Robust interaction of both EDEM2 and OS-9 with a non-glycosylated SHH variant indicates that the misfolded polypeptide backbone, rather than a glycan signature, functions as the predominant signal for recognition for ERAD. Notably, SHH-N278A is the first nonglycosylated substrate to require EDEM2 for recognition and targeting for ERAD. EDEM2 also interacts with calnexin and SEL1L, suggesting a potential avenue by which misfolded glycoproteins may be shunted towards SEL1L and ERAD rather than being released into the secretory pathway. Thus, ER lectins participate in the recognition and delivery of misfolded ER substrates differently in mammals, with an underlying mechanism distinct from that of S. cerevisiae.
    PLoS ONE 06/2014; 9(6):e92164. DOI:10.1371/journal.pone.0092164 · 3.23 Impact Factor
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    • "This strongly indicates that there indeed is a need for a higher capacity in the protein folding mechanism for efficient xylose fermentation. Whether this capacity is for regulation of disulphide bond formation [56], reduction of non-native disulphide bonds [57] or targeting unfolded glycoproteins for degradation [58] remains to be investigated. "
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    ABSTRACT: Efficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions. Four strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium. Increasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of the protein folding mechanism that were identified in transcription analysis of UPR related genes may also be limiting. This includes i) the transcription factor encoded by HAC1 ii) the activity of Pdi1p and iii) the requirement of free FAD during anaerobic growth.
    BMC Biotechnology 04/2014; 14(1):28. DOI:10.1186/1472-6750-14-28 · 2.59 Impact Factor
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