Defining the Glycan Destruction Signal for Endoplasmic Reticulum-Associated Degradation

Abstract

The endoplasmic reticulum (ER) must target potentially toxic misfolded proteins for retrotranslocation and proteasomal degradation while avoiding destruction of productive folding intermediates. For luminal proteins, this discrimination typically depends not only on the folding status of a polypeptide, but also on its glycosylation state. Two putative sugar binding proteins, Htm1p and Yos9p, are required for degradation of misfolded glycoproteins, but the nature of the glycan degradation signal and how such signals are generated and decoded remains unclear. Here we characterize Yos9p's oligosaccharide-binding specificity and find that it recognizes glycans containing terminal alpha1,6-linked mannose residues. We also provide evidence in vivo that a terminal alpha1,6-linked mannose-containing oligosaccharide is required for degradation and that Htm1p acts upstream of Yos9p to mediate the generation of such sugars. This strategy of marking potential substrates by Htm1p and decoding the signal by Yos9p is well suited to provide a proofreading mechanism that enhances substrate specificity.

Structural and Biochemical Basis of Yos9 Protein Dimerization and Possible Contribution to Self-association of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Degradation Ubiquitin-Ligase Complex

“…Previously, recombinantly expressed and refolded full-length Yos9 has been reported to run on a size exclusion column at an elution volume corresponding approximately to the theoretical size of the trimer (14). However, the shape of a molecule has a significant impact on its hydrodynamic radius, thereby potentially distorting the molecular mass estimate derived from size exclusion chromatography experiments.…”

“…Insoluble protein was purified and refolded as described in Ref. 14 Diffraction experiments were performed at beamline 14.1 (27) of the Helmholtz-Zentrum Berlin für Materialien und Energie and Freie Universität Berlin at BESSY. Selenomethionine derivative crystals were used for phasing by multiplewavelength anomalous diffraction.…”

“…In yeast, Htm1/Mnl1 was recently found to act as a mannosidase that catalyzes the last cleavage step before a glycan can function as a degradation signal (12,13). Yos9 acts downstream of Htm1/ Mnl1 by binding to those oligosaccharides that are produced by Htm1/Mnl1 cleavage, in particular to the terminal ␣-1,6-mannose residue (14,15).…”

Endoplasmic Reticulum-associated Degradation (ERAD) and Free Oligosaccharide Generation in Saccharomyces cerevisiae

“…2A, top, blue trace). Although amyloglucosidase cleaves ␣1,3-, ␣1,4-, and ␣1,6-glucose residues, it is possible that peaks a-e do not correspond to oligomers of ␣-linked glucose but rather correspond to ␤-glucans previously described to occur in yeast (Glc [3][4][5][6][7][8][9][10][11][12][13][14][15] (21)) because the amyloglucosidase preparation used in our studies is probably contaminated with ␤-glucanase (31). Another oligosaccharide pool was noted in both wild type and ams1⌬ cells but occurred in strikingly 3 I. Chantret and S. E. H. Moore, unpublished data.…”

Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption

“…The Htm1p mannosidase of this complex further trims down the glycan to the Man7GlcNAc2 form by removing the terminal mannose at the C-branch of the glycan, in order to create the substrate for HRD ligase-mediated glycoprotein degradation (Clerc et al 2009). The lectin Yos9p is part of this HRD ligase complex and recognizes glycans containing the now available terminal α1,6 linked mannose residues (Quan et al 2008). …”