Tyrosinase Maturation and Oligomerization in the Endoplasmic Reticulum Require a Melanocyte-specific Factor

Abstract

Tyrosinase is a glycoprotein responsible for the synthesis of melanin in melanocytes. A large number of mutations have been identified in tyrosinase, with many leading to its misfolding, endoplasmic reticulum (ER) retention, and degradation. Here we describe the folding and maturation of human tyrosinase (TYR) using an in vitro translation system coupled with ER-derived microsomes or with semipermeabilized cells, as an intact ER source. TYR remained misfolded as determined by its sensitivity to trypsin digestion and its persistent interaction with the ER resident lectin chaperones calnexin and calreticulin when produced in ER-derived microsomes or nonmelanocytic semipermeabilized cells. However, when TYR was translocated into semipermeabilized melanocytes, chaperone interactions were transient, maturation progressed to a trypsin-resistant state, and a TYR homodimer was formed. The use of semipermeabilized mouse melanocytes defective for tyrosinase or other melanocyte-specific proteins as the ER source indicated that proper TYR maturation and oligomerization were greatly aided by the presence of wild type tyrosinase and tyrosinase-related protein 1. These findings suggested that oligomerization is a step in proper TYR maturation within the ER that requires melanocyte-specific factors.

Carbohydrates act as sorting determinants in ER-associated degradation of tyrosinase

“…In support of this theory, previous studies show that mutant tyrosinase is misfolded (Francis et al, 2003;Halaban et al, 2000). Results of density gradient centrifugation indicated that control and mutant Tyr(C85S), and to some extent triglucosylated wild-type Tyr aggregated (Fig.…”

“…As a consequence, cytosolic components including proteasomes can be washed away and subsequently added back in a controlled manner. Here, SP-membranes were isolated by centrifugation and resuspended in RRL optimized for the proper maturation of tyrosinase (Francis et al, 2003). Tyrosinase mRNA was then added to the mixture to translate 35 S-labeled tyrosinase selectively.…”

“…Radioactive 35 S-labeled tyrosinase was translated for 1 hour in RRL in the presence of canine pancreas microsomes (MS) or semi-permeabilized (SP) melanocytes (Francis et al, 2003;Hebert et al, 1995). The standard translation mixture consisted of 1.75 µl MS or 3×10 5 semi-permeabilized cells, 1 µl RNasin, 13 µl RRL, 0.5 µl of 1 mM amino acid mixture minus methionine and cysteine, [ 35 S]Met/Cys (0.63 µCi), 3.2 mM DTT, 87 mM KCl, 2 mM oxidized glutathione (GSSG), and 500 ng mRNA.…”

“…Where indicated, the standard translation mixture was treated with 1 mM DMJ or DNJ for 10 minutes at 27°C prior to translation. SPmelanocytes were prepared from subconfluent melanocytes permeabilized with 20 µg/ml digitonin (Francis et al, 2003;Wilson et al, 1995).…”

Regulation of Tyrosinase Processing and Trafficking by Organellar pH and by Proteasome Activity

“…The known melanosomal proteins are involved in melanogenesis as catalytic and/or structural components and include TYR, TYRP1, DCT, MART-1, and gp100 (60,70). Although the processing and sorting of those proteins are not completely understood, they are known to be synthesized and translocated into the ER and eventually into the Golgi where their post-translational processing and glycosylation take place (36,71). Following that processing, they seem to take distinct routes to traffic to melanosomes, with the majority of melanosomal proteins predominantly going to stage II melanosomes, although that distribution is disrupted in amelanotic melanoma cells.…”