title: Identification of PNGase-dependent ERAD substrates in Saccharomyces cerevisiae.
abstract: Endoplasmic reticulum (ER)-associated degradation (ERAD) is a proteolytic pathway for handling misfolded or improperly assembled proteins that are synthesized in the ER. Cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans that are attached to ERAD substrates. While the critical roles of N-glycans in monitoring the folding status of carrier proteins in the ER lumen are relatively well understood, the physiological role of PNGase-mediated deglycosylation in the cytosol remained poorly understood. We report herein the identification of endogenous substrates for the cytoplasmic PNGase in Saccharomyces cerevisiae Using an isotope-coded glycosylation site-speciﬁc tagging (IGOT) method-based LC/MS analysis, 11 glycoproteins were specifically detected in the cytosol of PNGase-deletion cells (png1Δ). Among these molecules, at least five glycoproteins were clearly identified as ERAD substrates in vivo Moreover, four out of the five proteins were found to be either deglycosylated by PNGase in vivo or the overall degradation was delayed in a png1Δ mutant. Our results clearly indicate that the IGOT method promises to be a powerful tool for the identification of endogenous substrates for the cytoplasmic PNGase.
authors.affiliation: Glycometabolome Team, RIKEN-Max Planck Institute Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hosomi
authors.affiliation: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Fujita
authors.affiliation: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Tomioka
authors.affiliation: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaji
authors.affiliation: Glycometabolome Team, RIKEN-Max Planck Institute Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D002853:Chromatography, Liquid; D060746:Endoplasmic Reticulum-Associated Degradation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: ERAD; IGOT; PNGase; yeast