RAB11FIP1 interacts with the BLOC-1 complex to retrieve melanogenic proteins from the recycling pathway and a dominant negative mutation in RAB11FIP1 causes Hermanksy-Pudlak Syndrome Type 10 (HPS-10). A. R. Cullinane1, M. A. Merideth1, M. B. Datiles2, J. A. Curry1, N. F. Hansen3, J. K. Teer4, J. G. White5, J. C. Mullikin3,6, M. Huizing1, W. A. Gahl1 1) Medical Genetics Branch, NHGRI (NIH), Bethesda, MD; 2) NEI, NIH, Bethesda MD 20892, USA; 3) Comparative Genomics Analysis Unit, Cancer Genetics and Comparative Genomics Branch, NHGRI, NIH, Rockville, MD, USA; 4) Department of Biomedical Informatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa FL 33612, USA; 5) Department of Laboratory Medicine, University of Minnesota, Minneapolis, MN 55455, USA; 6) NIH Intramural Sequencing Center, NHGRI, NIH, Rockville MD 20852, USA.

   Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle (LRO) biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. There are currently 9 known genes that cause HPS; all of whose protein products function in the biogenesis of LROs. The Biogenesis of Lysosome related Organelle Complex 1 (BLOC-1) contains 8 subunits but relatively little is known about the intracellular function of the complex, although a role in endosomal protein sorting has been suggested. Using his-tagged BLOC-1 subunits expressed in HEK293 cells and mass spectroscopy, we discovered that RAB11FIP1 is a novel interacting protein of the BLOC-1 complex. RAB11FIP1 encodes a RAB11A interacting protein that homo-dimerizes to interact with RAB11A. A yeast-2-hybrid assay showed that the dysbindin subunit of BLOC-1 directly interacts with RAB11FIP1; this was confirmed by co-immunoprecipitation and confocal immunofluorescence microscopy in melanocytes. We now report a girl who had previously been screened for mutations in HPS1 through HPS6 and all the genes encoding the BLOC-1 complex. No mutations were found, although the patient had typical signs and symptoms of HPS and a cellular phenotype mimicking that of BLOC-1, i.e., increased plasma membrane cycling in melanocytes and endosomal accumulation of a melanogenic protein, TYRP1. Whole exome sequencing revealed a de novo heterozygous frameshift mutation in RAB11FIP1. The short protein fragment from this allele was expressed and interacted with the full-length protein, resulting in a dominant negative effect. Known cargos of the BLOC-1 complex in melanocytes are TYRP1 and ATP7A. How these cargos traffic to LROs was unknown, but we discovered that GFP-TYRP1 traffics to the plasma membrane, is endocytosed and only then is directed to LROs. We demonstrated that TYRP1 and ATP7A interact with the AP-1 complex, allowing this trafficking to occur. However, ATP7A appears to traffic directly to endocytic vesicles, where RAB11FIP1 and the BLOC-1 complex are required for retrieval to LROs. Taken together, these data suggest a function of the BLOC-1 complex in retarding protein recycling by forming a physical brake between early endosomes (through the BLOC-1 interactor, Syntaxin-13) and recycling endosomes (through the BLOC-1 interactor, RAB11FIP1). This would allow more time for proteins to be retrieved from the endosomal compartment (by the AP-1 complex) and directed to LROs.

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