BACKGROUND
hermansky-pudlak syndrome  is an autosomal recessive disorder of vesicle biogenesis resulting in the dysfunction of lysosome-related organelles such as melanosomes and platelet dense bodies  <cit> .
affected patients have oculocutaneous albinism presenting as congenital nystagmus, reduced visual acuity, and varying degrees of hypopigmentation of the skin, hair, and irides  <cit> .
in addition, a platelet storage pool deficiency, manifesting as absence of platelet dense bodies, causes loss of the secondary aggregation response  <cit> .
clinically, this results in easy bruising and epistaxis in childhood, prolonged bleeding during dental extractions and surgeries, and excessive menstrual and postpartum bleeding  <cit> .
some hps patients also develop granulomatous colitis or a fatal pulmonary fibrosis  <cit> .
to date, seven genes have been identified as causes of human hps subtypes , and other genes identified in mouse models of hps may also cause hps in humans  <cit> .
of the human subtypes, only hps- <dig>  results from mutations in a gene  with a known function.
ap3b <dig> codes for the β3a subunit of adaptor complex- <dig> , a coat protein that is involved in sorting transmembrane proteins to lysosomes and lysosome-related organelles  <cit> .
this recognized function of ap- <dig> supports the paradigm that all types of hps result from abnormal vesicle formation and/or trafficking.
each of the gene products of hps <dig> , hps <dig> , hps <dig> , hps <dig> , hps <dig> , and hps <dig>  is unique, although some hps proteins interact with each other in biogenesis of lysosome-related organelles complexes or blocs  <cit> .
the fact that these proteins have no homology to any known proteins, to each other, or to known functional domains makes them challenging candidates to investigate using in vitro methods.
in an attempt to understand the function of hps gene products, we focused on hps <dig>  a unique protein with a predicted clathrin-binding domain.
hps- <dig> patients, with mutations in the hps <dig> gene, have absent platelet dense bodies, mild to moderate hypopigmentation of skin and hair, iris transillumination, and patchy hypopigmentation of the fundus  <cit> .
the mouse ortholog of human hps- <dig>  cocoa, demonstrates similar features  <cit> .
the hps <dig> gene was identified by homozygosity mapping using hps patients from a central puerto rican genetic isolate  <cit> .
hps <dig> is located on chromosome 3q <dig> and has  <dig> exons and a  <dig> -bp open reading frame that codes for a 1004-amino acid protein.
the central puerto rican founder mutation in hps <dig> is a  <dig> -kb deletion encompassing exon  <dig> and its surrounding introns  <cit> .
the central puerto rican hps population is distinct from the hps isolate in northwest puerto rico, in which a founder mutation in the hps <dig> gene results in a severe form of hps  <cit> .
several non-puerto rican hps <dig> mutations, as well as an ashkenazi jewish founder mutation in hps <dig>  have now been identified  <cit> .
the human hps <dig> protein contains a predicted clathrin-binding motif  at residues 172– <dig> that conforms to the consensus sequence l. this consensus was determined by amino acid sequence alignment of the clathrin-binding regions in the beta subunits of adaptor proteins  <dig>   <dig>  and  <dig> , arrestin  <dig>  and amphiphysin i and ii  <cit> .
this 'clathrin box' is sufficient for binding to the amino terminal domain of the clathrin heavy chain  <cit> .
the structures of peptide complexes containing the clathrin-terminal domain and the clathrin-binding motifs of β-arrestin  <dig> and β3a have been determined by crystallography  <cit> .
both of these peptides bind, via their clathrin box motifs, to the same site in the clathrin heavy chain's terminal domain, with nearly identical bound conformations.
clathrin is the main component of protein coats that assist in the formation of vesicles budding from the trans-golgi network , plasma membrane, and endosomes.
several clathrin-associated proteins regulate the assembly of clathrin triskelions, composed of three heavy chains and three light chains, into the polyhedral cages that provide structure to intracellular vesicles.
some of these proteins and protein complexes are also involved in sorting cargo into vesicles and directing their transport within the cell  <cit> .
hps- <dig> disease results from deficiency of the clathrin binding protein β3a  <cit> , supporting a possible role for other hps proteins in clathrin binding.
hence, we investigated the clathrin binding function of the hps <dig> protein in melanocytes and fibroblasts.
RESULTS
several avenues of investigation were pursued to substantiate the predicted clathrin-binding activity of hps <dig>  these studies employed the techniques of immunoprecipitation, immunofluorescence, live cell imaging, and immunoelectron microscopy.
immunoprecipitation
whole cell lysates prepared from normal melanocytes and from hps <dig> null melanocytes, i.e., cells from a patient homozygous for the  <dig> -kb hps <dig> founder deletion  <cit> , exhibited approximately equal amounts of clathrin .
each whole cell lysate was then immunoprecipitated with polyclonal hps <dig> peptide antibodies.
clathrin, detected by antibodies to its heavy chain, co-immunoprecipitated with hps <dig> in the normal lysates but not in the hps <dig> null lysates .
these results were obtained using either of two different monoclonal anti-clathrin heavy chain antibodies.
immunofluorescence
at residues 172– <dig>  the hps <dig> protein has a predicted clathrin-binding motif  that is conserved in mouse and rat .
using site-directed mutagenesis, a gfp-hps3-delcbd construct was created in which the clathrin-binding motif was converted to non-conserved amino acids .
the interaction of clathrin-binding proteins with clathrin is a transient phenomena  <cit> .
to maximize the likelihood of observing the association of gfp-hps <dig> with clathrin by immunofluorescence, a 20°c temperature block was employed.
incubation at 20°c for  <dig> h was used to block trafficking out of the trans-golgi  <cit>  and followed by transfer to 37°c for  <dig> min to release the temperature block and resume normal trafficking.
normal melanocytes expressing wild-type gfp-hps <dig> or gfp-hps3-delcbd, so treated, were then fixed and stained with clathrin heavy chain antibodies.
cells expressing gfp-hps <dig> demonstrated partial co-localization with clathrin on one to several small vesicles in the perinuclear area .
in a representative experiment,  <dig> of  <dig> gfp-hps <dig> expressing melanocytes showed this co-localization.
in contrast,  <dig> of  <dig> cells exhibited co-localization of gfp-hps3-delcbd with clathrin .
live cell imaging
trafficking of acidic vesicles, stained with lysotracker red, was followed in live hps <dig> null fibroblasts expressing either gfp-hps <dig>  or gfp-hps3-delcbd .
following a 20°c temperature block and transfer to 37°c, acidic vesicles and gfp-hps <dig> clustered together in the perinuclear area .
gfp-hps <dig> transiently interacted with acidic vesicles and, in some cases , emerged together with them from the perinuclear area .
in contrast, following a 20°c block and transfer to 37°c, gfp-hps3-delcbd was predominantly localized in a punctate pattern in the periphery of the cell; no association of gfp-hps3-delcbd with acidic vesicles was observed .
immunoelectron microscopy
normal melanocytes were fixed approximately  <dig> h after transfection with gfp-hps <dig>  expression of the gfp-hps <dig> fusion protein was low at this time, thus minimizing aggregation and other artifacts that can occasionally be observed in overexpressing cells.
normal melanocytes transfected with gfp-hps3-delcbd were fixed later  because of the very low levels of expression obtained with this construct.
no aggregates were observed in the gfp-hps3-delcbd transfected cells.
gfp-hps <dig> localized predominantly to small  vesicles in the golgi region  anti-gfp immunogold particles; table 1) and the vast majority of these particles ) co-localized with clathrin .
at high magnification, gfp-hps <dig> labeling was demonstrated on well-defined clathrin-containing vesicles .
some of these small clathrin-containing vesicles were found near larger endosomal structures .
gfp-hps <dig> labeling was generally less abundant on large endosomal structures than on small vesicles  of anti-gfp immunogold particles found on endosomes) and fewer of these particles co-localized with clathrin ; figure  <dig>  table 1).
<dig> anti-gfp immunogold particles were counted in a total of  <dig> gfp-hps <dig> and  <dig> gfp-hps3-delcbd expressing cells
<dig> difference in number of gold particles per compartment, for gfp-hps3-delcbd compared with gfp-hps <dig>  is statistically significant by chi square analysis
<dig> difference in number of gold particles found in association with clathrin, for gfp-hps3-delcbd compared with gfp-hps <dig>  is statistically significant by chi square analysis
in contrast, gfp-hps3-delcbd expressing cells demonstrated very few anti-gfp immunogold particles on small vesicles in the golgi region ) and these were only rarely found in association with clathrin ; figure  <dig>  table 1).
the majority of anti-gfp immunogold particles localized to the cytoplasm of gfp-hps3-delcbd expressing cells ), compared with only 7%  in gfp-hps <dig> expressing cells .
these differences were statistically significant by chi square analysis .
double-labeling of anti-clathrin  and anti-gfp  in normal  melanocytes electroporated with gfp-hps3-delcbd .
gfp-hps3-delcbd  was largely cytoplasmic and distributed throughout the entire cell, from the perinuclear/golgi region  to the tips .
no co-localization of the two labels  was observed.
n=nucleus, g=golgi, e=endosomal structure, m=melanosome.
bar =  <dig> nm
no appreciable gfp-hps <dig> or gfp-hps3-delcbd labeling was observed on clearly identifiable golgi stacks .
as expected, clathrin labeling appeared much more abundant than gfp labeling in gfp-hps <dig> or gfp-hps3-delcbd expressing melanocytes.
similar amounts of gfp labeling per cell were detected in gfp-hps <dig> and gfp-hps3-delcbd expressing cells and no gfp labeling was observed in untransfected cells.
not all clathrin-containing membranes were associated with gfp-hps <dig>  but the majority of gfp-hps <dig> appeared to be associated with a clathrin-containing membrane.
discussion
hps <dig> is unusual among hps proteins in that it contains a predicted functional domain, i.e., a clathrin-binding motif  .
in this report, we describe evidence for the association of hps <dig> with clathrin.
we demonstrated that clathrin co-immunoprecipitates with endogenous hps <dig> in normal melanocytes; no clathrin was immunoprecipitated in the absence of hps <dig>  i.e., in hps <dig> null cells.
the necessity of the clathrin-binding domain in hps <dig> is supported by the partial co-localization of gfp-hps <dig> with clathrin only when this domain is present, as demonstrated by both fluorescence and immunoelectron microscopy studies.
furthermore, localization and trafficking of gfp-hps <dig> with acidic vesicles  depends upon an intact clathrin-binding domain.
the presence of a conserved clathrin-binding motif in hps <dig>  combined with the supportive data mentioned above, indicates that hps <dig> most likely binds clathrin directly.
we cannot, however, rule out the possibility that hps <dig> interacts indirectly with clathrin.
recent studies in mouse  <cit>  and human  <cit>  have shown that hps <dig>  interacts with hps <dig>  and hps <dig>  in the bloc- <dig> complex.
hence, hps <dig> may function as an essential component of a complex, such as bloc- <dig>  in which another member binds clathrin.
this seems unlikely, however, since hps <dig> and hps <dig> have no conserved clathrin-binding sequence motifs.
in our ultrastructural studies, gfp-hps <dig> was found primarily on small  clathrin containing vesicles in the perinuclear/golgi region of normal melanocytes.
mutation of the clathrin-binding domain of hps <dig> resulted in a largely cytoplasmic distribution of the fusion protein, suggesting that the clathrin-binding domain is necessary for the correct localization of hps <dig> and that clathrin recruits hps <dig> to small vesicles.
interestingly, no gfp-hps <dig> was seen on golgi stacks or on vesicles budding from the tgn, nor was it localized to large endosomal structures or mature melanosomes.
in melanocytes from hps <dig> deficient patients, dopa histochemistry  detected  <dig> nm dopa positive vesicles distributed throughout the cell  <cit> .
this was in contrast to the situation in normal melanocytes, in which the small dopa-positive vesicles were restricted to the golgi region.
a possible explanation is that hps <dig>  interacts with the small dopa-positive vesicles via its clathrin-binding domain, escorting them from the golgi region to premelanosomes for delivery of their contents.
in such a scenario, additional specialized accessory factors  would regulate vesicle targeting, budding and fusion events, clathrin coat assembly and disassembly, and interactions with the cytoskeleton.
future investigations should pursue the function of bloc- <dig> with the recognition that one of its components, hps <dig>  binds clathrin and may, therefore, bind to vesicles.
this understanding allows for hypotheses regarding the roles of hps <dig> and hps <dig> in bloc- <dig>  possible roles could include such functions as binding designated cargo, regulating conformational changes of the complex, or tethering proteins or vesicles for interactions with the bloc- <dig> complex as a whole.
CONCLUSIONS
hps <dig> 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 hps <dig> and is supported by immunoprecipitation, immunofluorescence, live cell imaging and immunoelectron microscopy data.

 hermansky-pudlak syndrome is an autosomal recessive disorder of vesicle biogenesis resulting in the dysfunction of lysosome-related organelles such as melanosomes and platelet dense bodies. affected patients have oculocutaneous albinism presenting as congenital nystagmus, reduced visual acuity, and varying degrees of hypopigmentation of the skin, hair, and irides. in addition, a platelet storage pool deficiency, manifesting as absence of platelet dense bodies, causes loss of the secondary aggregation response. clinically, this results in easy bruising and epistaxis in childhood, prolonged bleeding during dental extractions and surgeries, and excessive menstrual and postpartum bleeding. some hps patients also develop granulomatous colitis or a fatal pulmonary fibrosis. to date, seven genes have been identified as causes of human hps subtypes, and other genes identified in mouse models of hps may also cause hps in humans. of the human subtypes, only hps- results from mutations in a gene with a known function. ap3b codes for the β3a subunit of adaptor complex-, a coat protein that is involved in sorting transmembrane proteins to lysosomes and lysosome-related organelles. this recognized function of ap- supports the paradigm that all types of hps result from abnormal vesicle formation and/or trafficking. each of the gene products of hps, hps, hps, hps, hps, and hps is unique, although some hps proteins interact with each other in biogenesis of lysosome-related organelles complexes or blocs. the fact that these proteins have no homology to any known proteins, to each other, or to known functional domains makes them challenging candidates to investigate using in vitro methods. in an attempt to understand the function of hps gene products, we focused on hps a unique protein with a predicted clathrin-binding domain. hps- patients, with mutations in the hps gene, have absent platelet dense bodies, mild to moderate hypopigmentation of skin and hair, iris transillumination, and patchy hypopigmentation of the fundus. the mouse ortholog of human hps- cocoa, demonstrates similar features. the hps gene was identified by homozygosity mapping using hps patients from a central puerto rican genetic isolate. hps is located on chromosome 3q and has exons and a -bp open reading frame that codes for a 1004-amino acid protein. the central puerto rican founder mutation in hps is a -kb deletion encompassing exon and its surrounding introns. the central puerto rican hps population is distinct from the hps isolate in northwest puerto rico, in which a founder mutation in the hps gene results in a severe form of hps. several non-puerto rican hps mutations, as well as an ashkenazi jewish founder mutation in hps have now been identified. the human hps protein contains a predicted clathrin-binding motif at residues 172– that conforms to the consensus sequence l. this consensus was determined by amino acid sequence alignment of the clathrin-binding regions in the beta subunits of adaptor proteins and, arrestin and amphiphysin i and ii. this'clathrin box' is sufficient for binding to the amino terminal domain of the clathrin heavy chain. the structures of peptide complexes containing the clathrin-terminal domain and the clathrin-binding motifs of β-arrestin and β3a have been determined by crystallography. both of these peptides bind, via their clathrin box motifs, to the same site in the clathrin heavy chain's terminal domain, with nearly identical bound conformations. clathrin is the main component of protein coats that assist in the formation of vesicles budding from the trans-golgi network, plasma membrane, and endosomes. several clathrin-associated proteins regulate the assembly of clathrin triskelions, composed of three heavy chains and three light chains, into the polyhedral cages that provide structure to intracellular vesicles. some of these proteins and protein complexes are also involved in sorting cargo into vesicles and directing their transport within the cell. hps- disease results from deficiency of the clathrin binding protein β3a, supporting a possible role for other hps proteins in clathrin binding. hence, we investigated the clathrin binding function of the hps protein in melanocytes and fibroblasts.  several avenues of investigation were pursued to substantiate the predicted clathrin-binding activity of hps these studies employed the techniques of immunoprecipitation, immunofluorescence, live cell imaging, and immunoelectron microscopy. immunoprecipitation whole cell lysates prepared from normal melanocytes and from hps null melanocytes, i.e., cells from a patient homozygous for the -kb hps founder deletion, exhibited approximately equal amounts of clathrin. each whole cell lysate was then immunoprecipitated with polyclonal hps peptide antibodies. clathrin, detected by antibodies to its heavy chain, co-immunoprecipitated with hps in the normal lysates but not in the hps null lysates. these results were obtained using either of two different monoclonal anti-clathrin heavy chain antibodies. immunofluorescence at residues 172– the hps protein has a predicted clathrin-binding motif that is conserved in mouse and rat. using site-directed mutagenesis, a gfp-hps3-delcbd construct was created in which the clathrin-binding motif was converted to non-conserved amino acids. the interaction of clathrin-binding proteins with clathrin is a transient phenomena. to maximize the likelihood of observing the association of gfp-hps with clathrin by immunofluorescence, a 20°c temperature block was employed. incubation at 20°c for h was used to block trafficking out of the trans-golgi and followed by transfer to 37°c for min to release the temperature block and resume normal trafficking. normal melanocytes expressing wild-type gfp-hps or gfp-hps3-delcbd, so treated, were then fixed and stained with clathrin heavy chain antibodies. cells expressing gfp-hps demonstrated partial co-localization with clathrin on one to several small vesicles in the perinuclear area. in a representative experiment, of gfp-hps expressing melanocytes showed this co-localization. in contrast, of cells exhibited co-localization of gfp-hps3-delcbd with clathrin. live cell imaging trafficking of acidic vesicles, stained with lysotracker red, was followed in live hps null fibroblasts expressing either gfp-hps or gfp-hps3-delcbd. following a 20°c temperature block and transfer to 37°c, acidic vesicles and gfp-hps clustered together in the perinuclear area. gfp-hps transiently interacted with acidic vesicles and, in some cases, emerged together with them from the perinuclear area. in contrast, following a 20°c block and transfer to 37°c, gfp-hps3-delcbd was predominantly localized in a punctate pattern in the periphery of the cell; no association of gfp-hps3-delcbd with acidic vesicles was observed. immunoelectron microscopy normal melanocytes were fixed approximately h after transfection with gfp-hps expression of the gfp-hps fusion protein was low at this time, thus minimizing aggregation and other artifacts that can occasionally be observed in overexpressing cells. normal melanocytes transfected with gfp-hps3-delcbd were fixed later because of the very low levels of expression obtained with this construct. no aggregates were observed in the gfp-hps3-delcbd transfected cells. gfp-hps localized predominantly to small vesicles in the golgi region anti-gfp immunogold particles; table 1) and the vast majority of these particles ) co-localized with clathrin. at high magnification, gfp-hps labeling was demonstrated on well-defined clathrin-containing vesicles. some of these small clathrin-containing vesicles were found near larger endosomal structures. gfp-hps labeling was generally less abundant on large endosomal structures than on small vesicles of anti-gfp immunogold particles found on endosomes) and fewer of these particles co-localized with clathrin; figure table 1). anti-gfp immunogold particles were counted in a total of gfp-hps and gfp-hps3-delcbd expressing cells difference in number of gold particles per compartment, for gfp-hps3-delcbd compared with gfp-hps is statistically significant by chi square analysis difference in number of gold particles found in association with clathrin, for gfp-hps3-delcbd compared with gfp-hps is statistically significant by chi square analysis in contrast, gfp-hps3-delcbd expressing cells demonstrated very few anti-gfp immunogold particles on small vesicles in the golgi region ) and these were only rarely found in association with clathrin; figure table 1). the majority of anti-gfp immunogold particles localized to the cytoplasm of gfp-hps3-delcbd expressing cells ), compared with only 7% in gfp-hps expressing cells. these differences were statistically significant by chi square analysis. double-labeling of anti-clathrin and anti-gfp in normal melanocytes electroporated with gfp-hps3-delcbd. gfp-hps3-delcbd was largely cytoplasmic and distributed throughout the entire cell, from the perinuclear/golgi region to the tips. no co-localization of the two labels was observed. n=nucleus, g=golgi, e=endosomal structure, m=melanosome. bar = nm no appreciable gfp-hps or gfp-hps3-delcbd labeling was observed on clearly identifiable golgi stacks. as expected, clathrin labeling appeared much more abundant than gfp labeling in gfp-hps or gfp-hps3-delcbd expressing melanocytes. similar amounts of gfp labeling per cell were detected in gfp-hps and gfp-hps3-delcbd expressing cells and no gfp labeling was observed in untransfected cells. not all clathrin-containing membranes were associated with gfp-hps but the majority of gfp-hps appeared to be associated with a clathrin-containing membrane. discussion hps is unusual among hps proteins in that it contains a predicted functional domain, i.e., a clathrin-binding motif. in this report, we describe evidence for the association of hps with clathrin. we demonstrated that clathrin co-immunoprecipitates with endogenous hps in normal melanocytes; no clathrin was immunoprecipitated in the absence of hps i.e., in hps null cells. the necessity of the clathrin-binding domain in hps is supported by the partial co-localization of gfp-hps with clathrin only when this domain is present, as demonstrated by both fluorescence and immunoelectron microscopy studies. furthermore, localization and trafficking of gfp-hps with acidic vesicles depends upon an intact clathrin-binding domain. the presence of a conserved clathrin-binding motif in hps combined with the supportive data mentioned above, indicates that hps most likely binds clathrin directly. we cannot, however, rule out the possibility that hps interacts indirectly with clathrin. recent studies in mouse and human have shown that hps interacts with hps and hps in the bloc- complex. hence, hps may function as an essential component of a complex, such as bloc- in which another member binds clathrin. this seems unlikely, however, since hps and hps have no conserved clathrin-binding sequence motifs. in our ultrastructural studies, gfp-hps was found primarily on small clathrin containing vesicles in the perinuclear/golgi region of normal melanocytes. mutation of the clathrin-binding domain of hps resulted in a largely cytoplasmic distribution of the fusion protein, suggesting that the clathrin-binding domain is necessary for the correct localization of hps and that clathrin recruits hps to small vesicles. interestingly, no gfp-hps was seen on golgi stacks or on vesicles budding from the tgn, nor was it localized to large endosomal structures or mature melanosomes. in melanocytes from hps deficient patients, dopa histochemistry detected nm dopa positive vesicles distributed throughout the cell. this was in contrast to the situation in normal melanocytes, in which the small dopa-positive vesicles were restricted to the golgi region. a possible explanation is that hps interacts with the small dopa-positive vesicles via its clathrin-binding domain, escorting them from the golgi region to premelanosomes for delivery of their contents. in such a scenario, additional specialized accessory factors would regulate vesicle targeting, budding and fusion events, clathrin coat assembly and disassembly, and interactions with the cytoskeleton. future investigations should pursue the function of bloc- with the recognition that one of its components, hps binds clathrin and may, therefore, bind to vesicles. this understanding allows for hypotheses regarding the roles of hps and hps in bloc- possible roles could include such functions as binding designated cargo, regulating conformational changes of the complex, or tethering proteins or vesicles for interactions with the bloc- complex as a whole.  hps 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 hps and is supported by immunoprecipitation, immunofluorescence, live cell imaging and immunoelectron microscopy data. 
hps type- results from mutations in the hps gene, which encodes a amino acid protein of unknown function that contains a predicted clathrin-binding motif at residues 172– this association most likely occurs directly via a functional clathrin-binding domain in hps these results suggest a role for hps and its protein complex, bloc- in vesicle formation and trafficking. normal melanocytes expressing a gfp-hps fusion protein demonstrated partial co-localization of gfp-hps with clathrin following a 20°c temperature block. immunoelectron microscopy of normal melanocytes expressing gfp-hps showed co-localization of gfp-hps 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. these clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. hermansky-pudlak syndrome is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. hps associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region.
 hermansky-pudlak syndrome 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- results from mutations in the hps gene, which encodes a amino acid protein of unknown function that contains a predicted clathrin-binding motif at residues 172–  clathrin was co-immunoprecipitated by hps antibodies from normal but not hps null melanocytes. normal melanocytes expressing a gfp-hps fusion protein demonstrated partial co-localization of gfp-hps with clathrin following a 20°c temperature block. gfp-hps in which the predicted clathrin-binding domain of hps was mutated did not co-localize with clathrin under the same conditions. immunoelectron microscopy of normal melanocytes expressing gfp-hps showed co-localization of gfp-hps 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.  hps 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 hps these results suggest a role for hps and its protein complex, bloc- in vesicle formation and trafficking. 