Tyrosinase folding and copper loading in vivo: A crucial role for calnexinand alpha-glucosidase II

Tyrosinase is the key enzyme of melanin biosynthesis, It is a multiply glyc osylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. Th e 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 cha perone, 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 pro tein does not go through the normal secretory pathway and is targeted for d egradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase I I a critical role, but the association with calnexin is essential to promot e the correct folding which enables it to acquire copper. (C) 1999 Academic Press.