Cytoplasmic peptide:N-glycanase cleaves N-glycans on a carboxypeptidase Y mutant during ERAD in Saccharomyces cerevisiae.

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. GENERAL SIGNIFICANCE:This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged.

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