Abstract
Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.
Highlights
Tyrosinase is the rate-limiting enzyme involved in melanin biosynthesis [1, 2]
Soluble Tyrosinase Is Retained in the endoplasmic reticulum (ER) of Melanocytic and Non-melanocytic Cells—Constitutively synthesized only by melanocytes and melanoma cells, wild type tyrosinase is localized in specialized organelles named melanosomes
The major finding of this study is that the truncated tyrosinase is retained in the ER by a different set of chaperones than the previously reported oculocutaneous albinism (OCAI) mutants
Summary
Tyrosinase (monophenol, dihydroxy-phenylalanine: oxygen oxidoreductase; EC 1.14.18.1) is the rate-limiting enzyme involved in melanin biosynthesis [1, 2] This protein, consisting of a large catalytic lumenal domain that is anchored to the membrane by a C-terminal transmembrane domain and a short cytosolic tail [3], undergoes glycosylation prior to being subjected to the endoplasmic reticulum quality control [4]. As components of the quality control, were shown to interact transiently with the monoglucosylated N-glycans of the misfolded polypeptides [10, 11] These lectin chaperones engage the chains into the de-glucosylation/re-glucosylation cycle catalyzed by glucosidase II and glucosyltransferase, with the latter recognizing only incompletely, folded chains. Berson et al [21] have shown that the C-terminal truncation of both wild type and a temperaturesensitive mutant tyrosinase determined their ER retention
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