Conformation-dependent Post-translational Glycosylation of Tyrosinase

Tyrosinase, the rate-limiting enzyme in mammalian melanogenesis, is a copper-containing transmembrane glycoprotein. Tyrosinase undergoes a complex post-translational processing before reaching the melanosomal membrane. This processing involves N-glycosylation in several sites, including one located in the CuB copper binding site, movement from the endoplasmic reticulum (ER) to the Golgi, copper binding, and sorting to the melanosome. Aberrant processing is causally related to the depigmented phenotype of human melanomas. Moreover, some forms of albinism and several other pigmentary syndromes are considered ER retention diseases or trafficking defects. A critical step in tyrosinase maturation is the acquisition of an ER export-competent conformation recognized positively by the ER quality control system. However, the minimal structural requirements allowing exit from the ER to the Golgi have not yet been identified for tyrosinase or other melanosomal proteins. We addressed this question by analyzing the enzymatic activity and glycosylation pattern of mouse tyrosinase point mutants and chimeric constructs, where selected portions of tyrosinase were replaced by the homologous fragments of the highly similar tyrosinase-related protein 1. We show that a completely inactive tyrosinase point mutant lacking a critical histidine residue involved in copper binding is nevertheless able to exit from the ER and undergo further processing. Moreover, we demonstrate that tyrosinase displays at least two sites whose glycosylation is post-translational and most likely conformation-dependent and that a highly specific interaction involving the CuB site is essential not only for correct glycosylation but also for exit from the ER and enzymatic activity.