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

Abstract

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...

Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells

“…Both of these proteins are necessary for the processing and trafficking of tyrosinase and other proteins necessary for melanin synthesis in the melanosomes (31), and may also be important to maintain melanosomal pH (32). Similar to Slc15a4, the OCA2 polypeptide sequence contains dileucine motifs in its N-terminal region that are recognized by AP-3, which directs OCA2 to the mature melanosome (33).…”

TPC2 controls pigmentation by regulating melanosome pH and size

“…S5). This OCA2 loop was previously used to add other tags without affecting the protein trafficking and function (26). Providing OCA2 functions in chloride transport, we used the nonconductive V443I pore mutant to avoid any change MELOPS expression might cause on melanosomal pH (9).…”

Mechanisms of Protein Delivery to Melanosomes in Pigment Cells

“…These integral membrane proteins are recognized as melanosome cargoes in part by conventional cytoplasmically-exposed endosomal sorting signals (7, 19, 165, 168, 181, 189), likely functioning in concert with lumenal determinants (63, 168). The sorting signals in TYR, TYRP1 and OCA2 conform to the acidic dileucine-based consensus sequence of [D/E]XXXL[L/I] (14).…”