Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.
Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.
Branza-Nichita
N
N
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Petrescu
A J
AJ
Dwek
R A
RA
Wormald
M R
MR
Platt
F M
FM
Petrescu
S M
SM
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D003300:Copper / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology*; D008546:Melanoma, Experimental; D020460:Melanosomes / Q000378:metabolism; D051379:Mice; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D014407:Tumor Cells, Cultured; D000520:alpha-Glucosidases / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037281:Calnexin; D003300:Copper; D014442:Monophenol Monooxygenase; D000520:alpha-Glucosidases