Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen.
ER-associated degradation (ERAD) of glycoproteins depends on dual recognition of protein misfolding and remodeling of the substrate's N-linked glycans. After recognition, substrates are retrotranslocated to the cytosol for proteasomal degradation. To explore the directionality of this process, we fused a highly stable protein, DHFR, to the N or C terminus of the soluble ERAD substrate CPY* in yeast. Degradation of the C-terminal CPY*-DHFR fusion is markedly slowed and is accompanied by DHFR release in the ER lumen. Thus, folded lumenal domains can impede protein retrotranslocation. The ER lumenal protein Yos9p is required for both release of DHFR and degradation of multiple ERAD substrates. Yos9p forms a complex with substrates and has a sugar binding pocket that is essential for its ERAD function. Nonetheless, substrate recognition persists even when the sugar binding site is mutated or CPY* is unglycosylated. These and other considerations suggest that Yos9p plays a critical role in the bipartite recognition of terminally misfolded glycoproteins.
Bhamidipati
Arunashree
A
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.
Denic
Vladimir
V
Quan
Erin M
EM
Weissman
Jonathan S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D043402:Cathepsin A; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D006023:Glycoproteins; D037102:Lectins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C495027:Yos9 protein, S cerevisiae; D013762:Tetrahydrofolate Dehydrogenase; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae