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Identification of PNGase-dependent ERAD substrates in Saccharomyces cerevisiae
- Source :
- Biochemical Journal. 473:3001-3012
- Publication Year :
- 2016
- Publisher :
- Portland Press Ltd., 2016.
-
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.
- Subjects :
- 0301 basic medicine
Glycan
Saccharomyces cerevisiae Proteins
Glycosylation
Saccharomyces cerevisiae
Endoplasmic-reticulum-associated protein degradation
Biochemistry
Substrate Specificity
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Tandem Mass Spectrometry
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Molecular Biology
chemistry.chemical_classification
biology
Endoplasmic reticulum
Endoplasmic Reticulum-Associated Degradation
Cell Biology
biology.organism_classification
Cytosol
030104 developmental biology
chemistry
Cytoplasm
biology.protein
Glycoprotein
030217 neurology & neurosurgery
Chromatography, Liquid
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 473
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....993267518cd851bbc52b8dbf50d4524e