Sitaram, Anand
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Publication Mechanisms of Sorting and Trafficking for Melanosome Biogenesis as Revealed by Studies of the Human Pigment Cell Protein Oca2(2011-08-12) Sitaram, AnandVersion:1.0 StartHTML:0000000224 EndHTML:0000004814 StartFragment:0000002412 EndFragment:0000004778 SourceURL:file://localhost/Users/hastha82/Desktop/temporary%20desktop%20folder/MARKS%20LAB/THESIS/final%20changes.doc Certain cell types harbor specialized lysosome-related organelles (LROs) that derive from the endocytic system like conventional lysosomes but have unique functions. The coexistence of LROs and lysosomes in some cell types implies the existence of sorting mechanisms that divert resident cargo proteins to LROs. Based on the pigment-synthesizing melanosome in melanocytes as a model LRO and two melanosomal resident proteins as model cargoes, current models suggest that cargoes are sorted from early endosomes to melanosomes via one of two independent pathways mediated by the multisubunit complexes AP-3 or BLOC-1, each of which is defective in subtypes of the LRO biogenesis disease Hermansky-Pudlak Syndrome (HPS). An AP-3-related protein complex, AP-1, is thought to function in concert with BLOC-1. In this thesis, I assess the pigment-cell-specific putative transporter protein OCA2, as a third potential cargo protein with which to further dissect the relationships between AP-3, AP-1 and BLOC-1 in melanosomal transport. I first investigate the localization and site of action of OCA2. I use biochemical approaches in combination with site-directed mutagenesis and indirect immunofluorescence microscopic analysis of exogenously-expressed OCA2 in melanocytes to show that OCA2 is indeed a melanosome resident protein and does not function within the endoplasmic reticulum as has been suggested by other models. I show that melanosome localization is essential for OCA2 function and requires an acidic dileucine motif in the N-terminal cytoplasmic domain that can bind to both AP-3 and AP-1. Using site-directed mutagenesis in combination with yeast three hybrid assays and immunofluorescence microscopy analyses in melanocytes derived from mouse models of HPS and controls, I define the features of the OCA2 sorting signal that direct binding to AP-1 or AP-3 and show that OCA2 requires both AP-3 interaction and BLOC-1 for melanosomal localization. My results resolve a controversy regarding OCA2 localization, shed light on the interplay between AP-1 and AP-3 in melanosomal trafficking, and provide the first direct evidence for cooperation between BLOC-1 and AP-3 in trafficking to a LRO.