1. Subcellular Distribution of Tyrosinase and Tyrosinase-Related Protein-1: Implications for Melanosomal Biogenesis
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Sharon Pifko-Hirst, Denis J. Moran, Seth J. Orlow, and Raymond E. Boissy
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Tyrosinase ,Fluorescent Antibody Technique ,Skin Pigmentation ,Dermatology ,Biology ,Biochemistry ,Melanin ,03 medical and health sciences ,symbols.namesake ,Antigens, CD ,Lysosome ,medicine ,Tumor Cells, Cultured ,Humans ,Microscopy, Immunoelectron ,Molecular Biology ,Melanoma ,030304 developmental biology ,Melanosome ,0303 health sciences ,Membrane Glycoproteins ,Monophenol Monooxygenase ,030302 biochemistry & molecular biology ,Lysosome-Associated Membrane Glycoproteins ,Proteins ,Cell Biology ,Golgi apparatus ,Cell biology ,medicine.anatomical_structure ,Membrane protein ,Mutation ,symbols ,Melanocytes ,Oxidoreductases ,Percoll ,Biogenesis ,Subcellular Fractions - Abstract
Are tyrosinase, encoded at the albino locus, and tyrosinase-related protein-1 (TRP-1), encoded at the brown locus, similarly distributed in melanocytes? We determined the subcellular distribution of tyrosinase and TRP-1 using density fractionation of postnuclear supernatants from mouse melanoma cells of defined genotype followed by immunoblotting with specific antipeptide sera. In highly melanized cells, the majority of tyrosinase cosedimented on Percoll density gradients with visible melanin and with the peak of DOPA incorporation, confirming its presence predominantly in stage III-IV melanosomes. In contrast, the distribution of TRP-1 was limited to a less-dense melanosomal compartment, devoid of melanin. In amelanotic or minimally melanized cells, the majority of tyrosinase shifted into these lighter peaks. To explore a suspected relationship between lysosomes and melanosomes, we analyzed the distribution of lysosome-associated membrane protein-1 (LAMP-1). An overlap in the distribution of LAMP-1 and TRP-1 was demonstrated by immunomicroscopy and confirmed by immunoisolation. LAMP-1 was not present in the dense, melanin rich melanosomal peak on gradient analysis. TRP-1 from melanoma cells homozygous for the brown mutation is not fully glycosylated, is more rapidly degraded, and is restricted in its distribution compared to its wild-type counterpart. In these mutant cells, all melanosomal compartments contain LAMP-1. Our results demonstrate that in wild-type cells the majority of tyrosinase eventually localizes to stage III-IV melanosomes. TRP-1 is limited to a less dense melanosomal compartment that is also LAMP-1 positive. The existence of this compartment suggests that it may represent a common step in the biogenesis of melanosomes and lysosomes.
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