Abstract
In the endoplasmic reticulum (ER), N-linked glycans (N-glycans) function as signals to recruit the lectin chaperones involved in protein folding, quality control and ER-associated degradation. We undertook a systematic study of the four N-glycans of mutated carboxypeptidase yscY (CPY*) to determine whether there are positional differences between the glycans in ER-associated degradation. We constructed hypoglycosylated CPY* variants containing one, two or three N-glycans in various combinations and studied their degradation kinetics. We found that the four carbohydrate chains on CPY* are not equal in their signaling function: presence of the Asn368-linked glycan is necessary and sufficient for efficient degradation of CPY*. We also analysed the involvement of the ER lectins Htm1p and Cne1p (yeast calnexin) in the glycan-based recognition process with respect to number and position of N-glycans. We observed that Htm1p function depends on the presence of N-glycans in general but that there is no positional preference for a particular glycan. Cne1p, however, is selective with respect to substrate, and participates in the quality control only of some underglycosylated variants. For cases in which both lectins are involved, Cne1p and Htm1p play competing roles in targeting the substrate for degradation: loss of Cne1p accelerates degradation, whereas loss of Htm1p stabilizes the substrate.
Highlights
Secretory proteins enter the endoplasmic reticulum (ER) through the Sec61 translocation channel in an unfolded state
In the endoplasmic reticulum (ER), N-linked glycans (Nglycans) function as signals to recruit the lectin chaperones involved in protein folding, quality control and ERassociated degradation
We undertook a systematic study of the four N-glycans of mutated carboxypeptidase yscY (CPY*) to determine whether there are positional differences between the glycans in ER-associated degradation
Summary
Secretory proteins enter the endoplasmic reticulum (ER) through the Sec translocation channel in an unfolded state. In the ER, proteins are modified and folded to acquire their functional conformations (Haigh and Johnson, 2002; Rapoport et al, 1996). The folding process in the ER is controlled by a retention-based quality control system of ER-resident chaperones, protein disulfide isomerases (PDIs) and lectins. This system selectively distinguishes between properly folded proteins and incompletely folded, potentially cell-damaging conformers. Folded or orphan proteins are trapped in the ER, retrograde transported back into the cytosol and degraded by the ubiquitin-proteasome machinery (Brodsky and McCracken, 1999; Kostova and Wolf, 2003; Plemper et al, 1999; Sommer and Wolf, 1997; Wolf, 2004)
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