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 on 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 thesemolecules, at least 5 glycoproteins were clearly identified as ERAD substrates in vivo. Moreover, 4 out of the 5 proteins were found to be either deglycosylated by PNGase in vivo or the overall degradation was delayed in a png1 Δ 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.
- ©2016 The Author(s)
This is an Accepted Manuscript; not the final Version of Record. Archiving permitted only in line with the archiving policy of Portland Press Limited. All other rights reserved.