Carbohydrates act as sorting determinants in ER-associated degradation of tyrosinase.
The endoplasmic reticulum (ER) quality-control machinery maintains the fidelity of the maturation process by sorting aberrant proteins for ER-associated protein degradation (ERAD), a process requiring retrotranslocation from the ER lumen to the cytosol and degradation by the proteasome. Here, we assessed the role of N-linked glycans in ERAD by monitoring the degradation of wild-type (Tyr) and albino mutant (Tyr(C85S)) tyrosinase. Initially, mutant tyrosinase was established as a genuine ERAD substrate using intact melanocyte and semi-permeabilized cell systems. Inhibiting mannose trimming or accumulating Tyr(C85S) in a monoglucosylated form led to its stabilization, supporting a role for lectin chaperones in ER retention and proteasomal degradation. In contrast, ablating the lectin chaperone interactions by preventing glucose trimming caused a rapid disappearance of tyrosinase, initially due to the formation of protein aggregates, which were subsequently degraded by the proteasome. The co-localization of aggregated tyrosinase with protein disulfide isomerase and BiP, but not calnexin, supports an ER organization, which aids in protein maturation and degradation. Based on these studies, we propose a model of tyrosinase degradation in which interactions between N-linked glycans and lectin chaperones help to minimize tyrosinase aggregation and also target non-native substrates for retro-translocation and subsequent degradation.
Svedine
Sherri
S
Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, 710 North Pleasant Street, Amherst, MA 01003, USA.
Wang
Tao
T
Halaban
Ruth
R
Hebert
Daniel N
DN
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D037281:Calnexin / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005947:Glucose / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008544:Melanocytes / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D021381:Protein Transport
D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D037281:Calnexin; D000255:Adenosine Triphosphate; D014442:Monophenol Monooxygenase; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases; D005947:Glucose; D008358:Mannose