title: Cytoplasmic peptide:N-glycanase cleaves N-glycans on a carboxypeptidase Y mutant during ERAD in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected.
  
  
  METHODS
  We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity.
  
  
  RESULTS
  The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acid 400 and 404.
  
  
  CONCLUSIONS
  The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo.
  
  
  CONCLUSIONS
  This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged.
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: Glycometabolome Team, RIKEN-Max Planck Institute Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address: tsuzuki_gm@riken.jp.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D043402:Cathepsin A / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation; D006031:Glycosylation; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000502:physiology*; D011134:Polysaccharides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D043402:Cathepsin A; D046988:Proteasome Endopeptidase Complex; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: ER-associated degradation; Endoproteolysis; Saccharomyces cerevisiae; carboxypeptidase Y; peptide:N-glycanase