Multiple Endoplasmic Reticulum-associated Pathways Degrade Mutant Yeast Carboxypeptidase Y in Mammalian Cells

Abstract

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.

Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus

“…The fusion gene was then inserted downstream of the Aspergillus nidulans gpdA promoter in a plasmid containing a hygromycin resistance cassette, using the XbaI sites incorporated into each primer. The coding sequence of the Af-CPY gene was then modified by site-directed mutagenesis using primers 657 and 658 to create Af-CPY* (G264R), which creates the same point mutation that targets CPY* for degradation in S. cerevisiae (G255R) (23). The plasmid was linearized with KpnI and SacI and introduced into wt and ⌬hrdA protoplasts as an ectopically integrated expression construct using standard transformation procedures, as previously described (6).…”

Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

“…Indeed, recently multiple mechanisms have been reported to target different substrate proteins through distinct degradative pathways (4,27,44,50). Among the alternative degradation pathways, some are described as independent of calnexin but involving the ER chaperone calreticulin (4) or BiP (44).…”

“…Among the alternative degradation pathways, some are described as independent of calnexin but involving the ER chaperone calreticulin (4) or BiP (44). Another alternative pathway was found to be independent of cytosolic components but sensitive to either tyrosine phosphatase blockage or ER mannosidase I inhibition (27). Examples of substrates following such alternative pathways are mutant variants of ␣1-antitrypsin (4), carboxypeptidase Y (27), and tyrosinase (44,50).…”

“…Examples of substrates following such alternative pathways are mutant variants of ␣1-antitrypsin (4), carboxypeptidase Y (27), and tyrosinase (44,50). Other degradative pathways of glycoproteins may involve rhomboid intramembrane proteins (48) or a yet-uncharacterized proteasome-independent and nonlysosomal pathway (27).…”

Hepatic lipase maturation: a partial proteome of interacting factors

“…While Ub-mediated degradation is the most common pathway for ERAD (9,31,49), LPL degradation was unaffected by proteasomal inhibitors (23). Indeed, other examples indicate that multiple, nonproteasomal pathways of ERAD exist (50,51). However, in this study, we identified several proteins that suggest HL degradation is Ub mediated: CNX, Grp78, Grp94, and eEF1A are all established players in Ub-mediated degradation (9,31,33,49).…”