Multiple endoplasmic reticulum-associated pathways degrade mutant yeast carboxypeptidase Y in mammalian cells.
The degradation of misfolded and unassembled proteins by the endoplasmic reticulum (ER)-associated degradation (ERAD) has been shown to occur mainly through the ubiquitin-proteasome pathway after transport of the protein to the cytosol. Recent work has revealed a role for N-linked glycans in targeting aberrant glycoproteins to ERAD. To further characterize the molecular basis of substrate recognition and sorting during ERAD in mammalian cells, we expressed a mutant yeast carboxypeptidase Y (CPY*) in CHO cells. CPY* was retained in the ER in un-aggregated form, and degraded after a 45-min lag period. Degradation was predominantly by a proteasome-independent, non-lysosomal pathway. The inhibitor of ER mannosidase I, kifunensine, blocked the degradation by the alternate pathway but did not affect the proteasomal fraction of degradation. Upon inhibition of glucose trimming, the initial lag period was eliminated and degradation thus accelerated. Our results indicated that, although the proteasome is a major player in ERAD, alternative routes are present in mammalian cells and can play an important role in the disposal of both glycoproteins and non-glycoproteins.
Mancini
Roberta
R
Institute of Biochemistry, Swiss Federal Institute of Technology, Hoenggerberg, CH-8093 Zurich, Switzerland.
Aebi
Markus
M
Helenius
Ari
A
D000818:Animals; D016466:CHO Cells / Q000378:metabolism / Q000648:ultrastructure; D043402:Cathepsin A / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008361:Mannosidases / Q000037:antagonists & inhibitors; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D043402:Cathepsin A; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex