Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin.
BACKGROUND
Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172-176.


RESULTS
Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20 degrees C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution.


CONCLUSIONS
HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking.
Helip-Wooley
Amanda
A
Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA. ahwooley@mail.nih.gov
Westbroek
Wendy
W
Dorward
Heidi
H
Mommaas
Mieke
M
Boissy
Raymond E
RE
Gahl
William A
WA
Huizing
Marjan
M
D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism* / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D008544:Melanocytes / Q000737:chemistry / Q000166:cytology; D009154:Mutation; D011485:Protein Binding
D002352:Carrier Proteins; D002966:Clathrin; C432680:HPS3 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D049452:Green Fluorescent Proteins