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: 14.02). 06/2011; 42(6):782-93. DOI: 10.1016/j.molcel.2011.04.027
Source: PubMed


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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Available from: Robert Gauss, Jan 15, 2015
    • "ow - ing a cleavable signal sequence by PCR using RG394 ( 5′ - AAG TGG TTC GCA TCC TCG GTTT - 3′ ) and RG398 ( 5′ - CCC GAG CTC CAG GGG CCC CTG GAA CAG AAC TTC CAG ATG GTG ATG GTG ATG GTG ATG GTG ATG GTG GGT ACC CGC AAA GGC AGA ATG CGC - 3′ ) primers producing pRG101 . Finally , the PDI1 gene ( without the signal peptide ) was excised from pRG84 ( Gauss et al . 2011 ) by digestion with SacI and Xhol and introduced into pRG101 generating pRG105 . ER retention of sPdi1p is dis - rupted by the presence of two additional amino acids at the protein ' s C - terminus ( HDELLE ) . pRG143 , pRG144 , pRG145 , pRG146 : Truncated versions of the PDI1 gene were PCR - amplified from pRG84 using the following pri"
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    ABSTRACT: The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing.
    No preview · Article · Aug 2015 · Glycobiology
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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.
    Full-text · Article · Nov 2014 · Molecular Biology of the Cell
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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.
    Full-text · Article · Jun 2014 · PLoS ONE
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