Endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism

Abstract

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

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

“…This prediction was based in part on the colocalization of p with mutated Tyr in melan-c cells, which was assumed at the time to be situated in melanosomes, but to be enzymatically inactive. It has since been shown that the mutated Tyr in melan-c cells is in fact trapped in the ER and subsequently degraded (Halaban et al, 2000b). Indeed, the colocalization of Tyr and p in melan-c cells further strengthens our conclusions regarding the ER localization of p. However, it is still possible that a portion of total p may locate in other organelles.…”

“…Densitometric analysis revealed that approximately one-third of Tyr in melan-p1 cells is in this form. This form of Tyr was found to comigrate with Tyr from melan-c cells, derived from a mouse homozygous for point mutations in Tyr, which results in ER retention of Tyr (Halaban et al, 2000b).…”

“…Endoplasmic reticulum (ER) processing of Tyr requires the presence of the chaperone calnexin, which is thought to increase ER retention time for Tyr Toyofuku et al, 1999). The most common Tyr mutations are associated with ER retention of the protein, presumably the result of misfolding (Berson et al, 2000;Halaban et al, 2000b;Toyofuku et al, 2001). In addition, substrates DOPA and tyrosine promote Tyr exit from the ER and are associated with induction of Tyr activity and melanin synthesis in amelanotic melanoma cells (Halaban et al, 2000a).…”

Modulatory Proteins Can Rescue a Trafficking Defective Epileptogenic Nav1.1 Na+Channel Mutant

“…Trafficking defects are usually caused by the inability of the mutant protein to correctly fold and pass ER quality control system, and thus by its subsequent degradation (Cobbold et al, 2003;Sitia and Braakman, 2003). Incubation at permissive temperature typically rescues several of these misprocessed proteins (Denning et al, 1992;Payne et al, 1998;Halaban et al, 2000;Valdivia et al, 2002;Anderson et al, 2006). This has been interpreted as a kinetic effect because of more functional proteins able to correctly fold and escape the quality control of the ER because of the slowed kinetics of folding (Bernier et al, 2004).…”

Abnormal Acidification of Melanoma Cells Induces Tyrosinase Retention in the Early Secretory Pathway

“…Metabolic Labeling-Pulse-chase experiments were performed as described previously (8). Cells were pulse-labeled for 15 min with [ 35 S]Met/Cys (0.7 mCi/ml, EasyTag, PerkinElmer Life Sciences) in methionine/cysteine-free RPMI 1640 medium (Invitrogen) and either collected immediately or after chase incubation with non-radioactive medium (Ham's F-10 medium) for the indicated period of time.…”