title: The cotranslational maturation of the type I membrane glycoprotein tyrosinase: the heat shock protein 70 system hands off to the lectin-based chaperone system.
abstract: The maturation of eukaryotic secretory cargo initiates cotranslationally and cotranslocationally as the polypeptide chain emerges into the endoplasmic reticulum lumen. Here, we characterized the cotranslational maturation pathway for the human type I membrane glycoprotein tyrosinase. To recapitulate the cotranslational events, including glycosylation, signal sequence cleavage, chaperone binding, and oxidation, abbreviated transcripts lacking a stop codon were in vitro translated in the presence of semipermeabilized melanocyte membranes. This created a series of ribosome/translocon-arrested chains of increasing lengths, simulating intermediates in the cotranslational folding process. Initially, nascent chains were found to associate with the heat shock protein (Hsp) 70 family member BiP. As the nascent chains elongated and additional glycans were transferred, BiP binding rapidly decreased and the lectin-based chaperone system was recruited in its place. The lectin chaperone calnexin bound to the nascent chain after the addition of two glycans, and calreticulin association followed upon the addition of a third. The glycan-specific oxidoreductase ERp57 was cross-linked to tyrosinase when calnexin and calreticulin were associated. This timing coincided with the formation of disulfide bonds within tyrosinase and the cleavage of its signal sequence. Therefore, tyrosinase maturation initiates cotranslationally with the Hsp70 system and is handed off to the lectin chaperone system that first uses calnexin before calreticulin. Interestingly, divergence in the maturation pathways of wild-type and mutant albino tyrosinase can already be observed for translocon-arrested nascent chains.
authors.affiliation: Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.forename: Robert
authors.initials: R
authors.lastname: Daniels
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Hebert
mesh_terms: D000818:Animals; D037281:Calnexin / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D045744:Cell Line, Tumor; D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005165:Factor V / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007535:Isomerases / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000502:physiology; D021381:Protein Transport; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels
chemical_list: D037282:Calreticulin; D004220:Disulfides; D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D037102:Lectins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D037281:Calnexin; D005165:Factor V; D014442:Monophenol Monooxygenase; D007535:Isomerases; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human