Tyrosinase is a melanocyte-specific enzyme critical for the synthesis of melanin, a process normally restricted to a post-Golgi compartment termed the melanosome. Loss-of-function mutations in tyrosinase are the cause of oculocutaneous albinism, demonstrating the importance of the enzyme in pigmentation. In the present study, we explored the possibility that trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi apparatus and beyond is disrupted. Toward this end, we analyzed the common albino mouse mutation Tyr(C85S), the frequent human albino substitution TYR(T373K), and the temperature-sensitive tyrosinase TYR(R402Q)͞Tyr(H402A) found in humans and mice, respectively. Intracellular localization was monitored in albino melanocytes carrying the native mutation, as well as in melanocytes ectopically expressing green fluorescent protein-tagged tyrosinase. Enzymatic characterization of complex glycans and immunofluorescence colocalization with organelle-specific resident proteins established that all four mutations produced defective proteins that were retained in the ER. TYR(R402Q)͞Tyr(H402A) Golgi processing and transport to melanosomes were promoted at the permissive temperature of 32°C, but not at the nonpermissive 37°C temperature. Furthermore, evidence of protein misfolding was demonstrated by the prolonged association of tyrosinase mutants with calnexin and calreticulin, known ER chaperones that play a key role in the quality-control processes of the secretory pathway. From these results we concluded that albinism, at least in part, is an ER retention disease.calnexin ͉ protein folding ͉ quality control
In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4 -7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.
Tyrosinase is essential for pigmentation and is a source of tumor-derived antigenic peptides and cellular immune response. Wild type tyrosinase in melanoma cells and certain albino mutants in untransformed melanocytes are targeted to proteolytic degradation by the 26 S proteasome due to retention of the misfolded protein in the endoplasmic reticulum and its subsequent retranslocation to the cytosol. Here, we demonstrate that the substrates DOPA and tyrosine induced in melanoma cells a transition of misfolded wild type tyrosinase to the native form that is resistant to proteolysis, competent to exit the endoplasmic reticulum, and able to produce melanin. Because the enzymatic activity of tyrosinase is induced by DOPA, we propose that proper folding of the wild type protein, just like mutant forms, is tightly linked to its catalytic state. Loss of pigmentation, therefore, in tyrosinase-positive melanoma cells is a consequence of tumor-induced metabolic changes that suppress tyrosinase activity and DOPA production within these cells.
The endoplasmic reticulum (ER) quality-control machinery maintains the fidelity of the maturation process by sorting aberrant proteins for ER-associated protein degradation (ERAD), a process requiring retrotranslocation from the ER lumen to the cytosol and degradation by the proteasome. Here, we assessed the role of N-linked glycans in ERAD by monitoring the degradation of wild-type (Tyr) and albino mutant (Tyr(C85S)) tyrosinase. Initially, mutant tyrosinase was established as a genuine ERAD substrate using intact melanocyte and semi-permeabilized cell systems. Inhibiting mannose trimming or accumulating Tyr(C85S) in a monoglucosylated form led to its stabilization, supporting a role for lectin chaperones in ER retention and proteasomal degradation. In contrast, ablating the lectin chaperone interactions by preventing glucose trimming caused a rapid disappearance of tyrosinase, initially due to the formation of protein aggregates, which were subsequently degraded by the proteasome. The co-localization of aggregated tyrosinase with protein disulfide isomerase and BiP, but not calnexin, supports an ER organization, which aids in protein maturation and degradation. Based on these studies, we propose a model of tyrosinase degradation in which interactions between N-linked glycans and lectin chaperones help to minimize tyrosinase aggregation and also target non-native substrates for retro-translocation and subsequent degradation.
Protein modifications such as ubiquitination and phosphorylation commonly serve as sorting tags that control the trafficking and stability of a protein within the cytosol. In recent years, N-linked glycans have emerged as key protein modifications for eukaryotic secretory proteins. These modifications support the recruitment of molecular chaperones and sorting receptors, which recognize specific glycoforms. Therefore, glycanases and carbohydrate transferases work in concert with lectin chaperones and receptors to aid in the maturation and quality control of glycoproteins.
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