Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.

Rosemblat, Susana; Durham‐Pierre, Donna; Gardner, James A.; Nakatsu, Yoshimichi; Brilliant, Murray H.; Orlow, Seth J.

doi:10.1073/pnas.91.25.12071

Cited by 139 publications

(117 citation statements)

References 24 publications

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“…Using Percoll and sucrose gradient fractionation in combination with immunofluorescence microscopy, we show that the ER contains a significant amount of p protein. This is in contrast to our previous prediction (Rosemblat et al, 1994) that p is a melanosomal protein. 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.…”

Section: Discussioncontrasting

confidence: 55%

“…This prompted us to reexamine the location of p. It was generally thought that p was located in melanosomes (Rosemblat et al, 1994;Donatien and Orlow, 1995). However, a combination of data from sucrose gradient and Percoll gradient fractionation as well as immunofluorescence microscopy reveals that the ER contains a significant amount of p protein.…”

Section: The Majority Of P Is Located In the Ermentioning

confidence: 99%

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Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

Chen

Manga

Orlow

2002

MBoC

127

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The processing of tyrosinase, which catalyzes the limiting reaction in melanin synthesis, was investigated in melan-p1 melanocytes, which are null at the p locus. Endoglycosidase H digestion showed that a significant fraction of tyrosinase was retained in the endoplasmic reticulum. This retention could be rescued either by transfection of melan-p1 cells with an epitope-tagged wild-type p transcript or by treatment with either bafilomycin A1 or ammonium chloride. We found that the endoplasmic reticulum contains a significant amount of p protein, thus supporting a role for p within this compartment. Using immunofluoresence, we showed that most mature full-length tyrosinase in melan-p1 cells was located in the perinuclear area near the Golgi, in contrast to its punctate melanosomal pattern in wild-type melanocytes. Expression of p in melan-p1 cells restored tyrosinase to melanosomes. Triton X-114 phase separation revealed that an increased amount of tyrosinase was proteolyzed in melan-p1 cells compared with wild-type melanocytes. The proteolyzed tyrosinase was no longer membrane bound, but remained enzymatically active and a large proportion was secreted into the culture medium of melan-p1 cells. We conclude that p regulates posttranslational processing of tyrosinase, and hypopigmentation in melan-p1 cells is the result of altered tyrosinase processing and trafficking.

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Section: Discussioncontrasting

confidence: 55%

Section: The Majority Of P Is Located In the Ermentioning

confidence: 99%

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

Chen

Manga

Orlow

2002

MBoC

127

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“…214, 218, and 273) and additional sites elsewhere, but mouse OCA2 was suggested to be nonglycosylated based on lack of a mobility shift in tunicamycin-treated cells (Rosemblat et al, 1994). We thus tested whether human OCA2 was in fact N-glycosylated using endoglycosidase treatment in a metabolic pulse/chase and immunoprecipitation experiment.…”

Section: Molecular Biology Of the Cell 1466mentioning

confidence: 99%

“…OCA2 was originally thought to localize predominantly to mature melanized melanosomes based on subcellular fractionation of melanocytes (Rosemblat et al, 1994), poor extraction by detergent from melanized melanocytes relative to nonmelanized melanocytes (Donatien and Orlow, 1995), and interpretation of results from confocal immunofluorescence microscopy (IFM) analyses (Toyofuku et al, 2002). Tyrp1-containing compartments in melanocytes from OCA2-deficient mice are less acidic than in wild-type melanocytes, suggesting that OCA2 not only localizes to melanosomes but also modulates their pH (Puri et al, 2000), although this interpretation is disputed because Tyrp1 does not localize to melanosomes properly in OCA2-deficient melanocytes (Manga et al, 2001).…”

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confidence: 99%

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

Sitaram

Piccirillo

Palmisano

et al. 2009

MBoC

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Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steadystate lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes. INTRODUCTIONMelanin pigments are synthesized by specialized cell types, including dermal and epidermal melanocytes and retinal pigment epithelial cells, within unique organelles known as melanosomes . Melanosomes are members of a class of tissue-specific "lysosome-related organelles" characterized by an acidic lumenal pH and the presence of some lysosomal proteins (Dell'Angelica et al., 2000;Griffiths, 2002). Among lysosome-related organelles, melanosomes represent a subclass that coexists with bona fide lysosomes in their host cells . Melanosomes are distinguished from lysosomes by the presence of cell-type-specific cargo proteins that confer unique functional and morphological properties. How these cargo proteins are diverted from traditional lysosomes and delivered to and maintained within melanosomes is incompletely understood, and the degree of cargo cross-talk between melanosomes and lysosomes is not known.Melanosomes undergo a program of maturation within melanocytes by the ordered delivery of cargoes to nascent melanosomes via specialized trafficking pathways . Some components of the melanosomal trafficking machinery are known, largely from analyses of the sorting of well-characterized cargo proteins, such as the melanogenic enzyme tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1), in both wild-type melanocytes and melanocytes derived from patients or mouse models of genetic hypopigmentary diseases such as Hermansky-Pudlak Syndrome (HPS) (Di Pietro and Dell'Angelica, 2005;Wei, 2006). Tyr and Tyrp1 contain cytoplasmic a...

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“…These include tyrosinase (TYR), the tyrosinase-related proteins-1 and -2 (TYRP1͞TRP1 and DCT͞TRP2, respectively; refs. 1-3), GP100͞Pmel17͞silver (4)(5)(6), and Pink (7)(8)(9). In addition, another protein, termed MART1 (melanoma antigen recognized by T lymphocytes), has been cloned that localizes in melanosomes (10,11), but its function there remains unclear.…”

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confidence: 99%

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

Kushimoto

Basrur

Valencia

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

214

263

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Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.pigment ͉ melanin ͉ tyrosinase ͉ melanoma M ore than 95 distinct genes that play direct or indirect roles in mammalian pigmentation have been identified. Many of these genes encode proteins that are localized in melanosomes, specialized pigment organelles produced only by melanocytes. These gene products alter the quality or quantity of melanin produced and͞or the processing and distribution of melanosomes. The known melanosomal proteins are involved in melanogenesis as catalytic and͞or structural components. These include tyrosinase (TYR), the tyrosinase-related proteins-1 and -2 (TYRP1͞TRP1 and DCT͞TRP2, respectively; refs.

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Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.

Abstract: The pink-eyed dilution (p) locus In the mouse is critical to manoge; mutation in the homologous locus in humans, P, are a cause of type I oculocutaneous albm.

Cited by 139 publications

References 24 publications

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

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