We reasoned that the direct measurement of melanosomal metabolites would enable a better understanding of the function of melanosomal proteins in cells. Our current understanding of melanin synthesis has been in large part driven by the study of cell-free melanin synthesis reactions and the chemical degradation of isolated melanins 5, 6. Many of these proteins have been implicated in human pigment variation, but in many cases their biochemical functions remain unknown or unclear 1, 2. While in cell-free systems tyrosinase, tyrosine, and oxygen are sufficient to produce simple melanin polymers, in vivo additional melanosomal proteins influence the chemical composition and quantity of the melanin synthesized. Within melanosomes, tyrosinase catalyzes the oxidation of tyrosine to dopaquinone, which is then further oxidized and polymerized into a melanin pigment 5, 6. Pigment producing cells like melanocytes synthesize melanin in membrane-bound, lysosome-related organelles (LRO) called melanosomes. Thus, MFSD12 is an essential component of the long-sought cysteine importer for melanosomes and lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal storage disease caused by inactivation of the lysosomal cystine exporter CTNS (Cystinosin) 7– 9. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes, and, in non-pigmented cells, lysosomes. We find that MFSD12 is required to maintain normal levels of cystine, the oxidized dimer of cysteine, in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation 5, 6. We use it to study MFSD12, a transmembrane protein of unknown molecular function that when suppressed causes darker pigmentation in mice and humans 3, 4. Here, we describe the MelanoIP method for rapidly isolating melanosomes and profiling their labile metabolite contents. Many of these encode proteins that localize to the melanosome, the lysosome-related organelle that synthesizes pigment, but have unclear functions 1, 2. Dozens of genes contribute to the vast variation in human pigmentation.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |