Human Molecular Genetics, 2000, Vol. 9, No. 3 375-385
© 2000 Oxford University Press
The Hermansky–Pudlak syndrome (HPS) protein is part of a high molecular weight complex involved in biogenesis of early melanosomes
Human Medical Genetics Program, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, B-161, Denver, CO 80262, USA and 1Section Recherche, Institut Curie, Paris 75005, France
Received 28 September 1999; Revised and Accepted 1 December 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder in which oculocutaneous albinism, bleeding tendency and a ceroid-lipofuscin lysosomal storage disease result from defects of multiple cytoplasmic organelles: melanosomes, platelet dense granules and lysosomes. The HPS polypeptide, a 700 amino acid protein which is unrelated to any known proteins, is likely to be involved in the biogenesis of these different organelles. Here, we show that HPS is a non-glycosylated, non-membrane protein which is a component of two distinct high molecular weight complexes. In non-melanotic cells the HPS protein is contained almost entirely in an ~200 kDa complex that is widely distributed throughout the cytosol. In melanotic cells the HPS protein is partitioned between this cytosolic complex and a >500 kDa complex that appears to consist of the ~200 kDa complex in association with membranous components. Subcellular fractionation, immunofluorescence and immunoelectron microscopy studies indicate that the membrane-associated HPS complex of melanotic cells is associated with tubulovesicular structures, small non-coated vesicles, and nascent and early-stage melanosomes. These findings suggest that the HPS complex is involved in the biogenesis of early melanosomes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hermansky–Pudlak syndrome (HPS; MIM 203300) is a rare autosomal recessive disorder characterized by tyrosinase-positive oculocutaneous albinism, a tendency to bleed and a poorly defined ceroid-lipofuscin lysosomal storage disease, with frequent fatal complications (1; for reviews see refs 2,3). At the cellular level, HPS is associated with defects of multiple cytoplasmic organelles: melanosomes, platelet dense granules and lysosomes. HPS has thus been thought to result from a defect of a protein required for the biogenesis, structure or function of these different membrane-bound organelles (2,4).
We previously described positional cloning of the HPS gene (5) and identification of a number of pathological gene mutations in patients with HPS (5–7). Two major HPS mRNA isoforms, the result of alternative splicing of exon 9 encoding amino acids 290–313 (5,8), are coordinately expressed in many or all tissues and cell types (5) and encode major and minor HPS protein isoforms of 79.3 and 75.9 kDa, respectively. Despite cloning of the gene, clues to the biological function of the HPS protein have been elusive. HPS has no apparent homology to any other known proteins and contains no major protein motifs (5). Previous computer analysis of the HPS amino acid sequence predicted two potential transmembrane domains, two potential N-glycosylation sites, and a putative C-terminal melanosomal localization signal (5), although this last is not conserved in the mouse (9) and rat (J. Oh and R.A. Spritz, unpublished data), and therefore its significance now seems doubtful. Evolutionary comparisons (9) and mutational analyses (5–7,10) of the HPS gene have likewise failed to highlight conserved or critical segments of the coding region that might reflect functionally significant portions of the protein.
We have carried out a series of studies of the biochemical properties and cell biology of the HPS protein to begin to define its role in organellar biogenesis. We developed antibodies to human HPS polypeptide segments and used these to study biochemical characteristics and intracellular compartmentalization of the HPS protein in cultured human cells. The HPS protein is non-glycosylated and is not an integral membrane protein. In non-melanotic cells the HPS protein is contained in an ~200 kDa complex that is widely distributed throughout the cytosol. However, in melanotic cells about half of the HPS protein is part of a >500 kDa membrane-associated complex that is associated with small, non-coated vesicles and tubulovesicular structures in the peri-Golgi region, and with nascent and early-stage melanosomes. Our findings suggest that the HPS protein is part of a ubiquitous cytosolic complex that in melanotic cells becomes transiently but specifically associated with membranous components of melanosomal progenitors during early stages of organellogenesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies to the human HPS protein
Rabbit polyclonal antisera and mouse monoclonal antibodies (mAbs) were raised against a mixture of two segments of the human HPS protein, residues 1–463 and 462–700, which together span the entire 700 amino acid HPS polypeptide. As shown in Figure 1a, in western blots of whole cell extracts of normal human lymphoblastoid cells, affinity-purified antiserum hHPS504 detected major and minor bands that are likely to correspond to the major and minor HPS protein isoforms (79.3 and 75.9 kDa, respectively) that result from alternative splicing of HPS mRNA (5,8). Both bands were absent in western blots of extracts of lymphoblastoid cells from an HPS patient homozygous for a codon 491–496 frameshift, consistent with the lack of stable HPS mRNA associated with this mutation (11). Identical results were obtained using extracts of normal versus HPS-mutant fibroblasts (data not shown).
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We obtained similar results using 21 different mouse mAbs generated against the human HPS protein. For example, as shown in Figure 1a, mAb hHPS5 identified the major and minor HPS protein isoforms in western blots of extracts of FME melanoma cells, versus no bands in the extract of lymphoblastoid cells from an HPS patient. Again, identical results were obtained using extracts of normal versus HPS-mutant fibroblasts (data not shown).
mAb hHPS5 was also useful for indirect immunofluorescence microscopy. As shown in Figure 1b, hHPS5 detected strong signal throughout the cytoplasm of normal human fibroblasts, but no signal in HPS-mutant fibroblasts. Accordingly, mAb hHPS5 was used for all western blots and microscopic analyses described here.
Cell fractionation analysis
To define the subcellular distribution of the HPS protein, we carried out fractionation of various human cell types and assayed the fractions by western blotting using antibodies specific to various subcellular components. In all cell types studied, the HPS protein was detected only in cytoplasmic fractions, in contrast to nuclear protein Mre11, which we detected only in the nuclear pellet fraction (data not shown); HPS is thus a cytoplasmic protein. As shown in Figure 2a, in fractionated FME melanoma cells we detected the HPS protein in the post-nuclear 1000 g cytoplasmic supernatant (PNS). On further fractionation, the HPS protein partitioned in roughly equal proportions into the 10 000 g cytoplasmic pellet ‘large granule fraction’ (LGF; contains lysosomes and all stages of melanosomes) and the subsequent 100 000 g supernatant cytosolic (S100) fraction; a much smaller amount was detected in the 100 000 g pellet (P100; contains endosomes and small vesicles). As expected, tyrosinase, a melanosomal membrane protein, partitioned principally into the LGF and P100 fractions.
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Surprisingly, we obtained rather different results in analyses of non-melanotic cells (lymphoblastoid cells and fibroblasts). As shown in Figure 2b, in lymphoblastoid cells we detected the HPS protein principally in the S100 and to a lesser extent in the P100 fraction, but not in the LGF, which would contain the lysosomes. Indeed, LAMP1, a membrane marker principally for lysosomes and endosomes (12), was detected only in the LGF and to a lesser extent the P100 fractions. Identical data were obtained for fibroblasts (data not shown). Thus, in melanotic cells the HPS protein is distributed largely between the cytosol and a large granule/organellar compartment, with a much smaller amount in the endosomal/small vesicle compartment, whereas in non-melanotic cells the HPS protein is absent from the large granule/organellar compartment. This difference may well reflect the fact that melanocytes are involved in the pathology of HPS, whereas fibroblasts and lymphocytes are not.
The HPS protein is not N-glycosylated
Previous computer motif analysis of the HPS polypeptide sequence had predicted two potential N-glycosylation sites and two potential transmembrane domains (5), characteristics that might be expected of an integral lysosomal/melanosomal protein transported via a trans-Golgi network (TGN)-endosomal trafficking system. To determine whether or not the HPS protein is N-glycosylated we carried out two different assays. First, we enzymatically treated FME melanoma cell extracts with endoglycosidase H, which specifically cleaves N-linked hybrid or high-mannose oligosaccharide chains (13). Second, we cultured FME melanoma cells in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation (14), followed by western blot analysis of cell extracts. As shown in Figure 3a, endoglycosidase H cleavage had no apparent effect on the HPS protein on western blot analysis, whereas it resulted in marked reduction of apparent molecular mass of LAMP1, due to cleavage of its N-linked oligosaccharide chains. Similarly, as shown in Figure 3b, culture of melanoma cells with tunicamycin did not reduce the apparent amount of HPS protein, whereas the amount of LAMP1 protein was greatly diminished, as expected, the result of degradation due to its reduced glycosylation (15). Together, these results indicate that the HPS protein is not N-glycosylated in melanoma cells.
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HPS is not an integral membrane protein, but is partially membrane associated
To determine whether or not the HPS protein is an integral membrane protein we carried out phase separation of lymphoblastoid and melanoma cell fractions using Triton X-114, a non-ionic detergent that segregates hydrophilic proteins into the aqueous phase and amphiphilic integral membrane proteins into the detergent phase (16,17). As shown in Figure 4, the HPS protein partitioned exclusively into the soluble phases of the LGF (LGFSol) and corresponding 10 000 g supernatant fractions (S10Sol) of FME melanoma cells, and into the S10Sol of lymphoblastoid cells. In contrast, tyrosinase, a melanosomal membrane protein, partitioned almost completely into the detergent phase of the LGF (LGFMem) and to a much lesser extent the detergent phase of the 10 000 g supernatant fraction (S10Mem) of FME melanoma cells. Likewise, LAMP1, a lysosomal membrane protein, partitioned almost completely into the LGFMem and S10Mem phases of lymphoblastoid cells (Fig. 4). These data thus indicate that the HPS polypeptide is not an integral membrane protein.
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To determine whether the HPS protein might nevertheless be membrane associated, we tested the effect of Tris–HCl, which intercalates into membranes and can disrupt membrane–protein interactions (18), on the relative distribution of LGF-associated versus cytosolic HPS protein. As described above, HPS protein from FME melanoma cells lysed in buffer containing 25 mM HEPES pH 6.8 partitioned almost equally into the LGF and S100 cytosolic fractions (Fig. 2a). In contrast, HPS protein from FME melanoma cells lysed in buffer containing 50 mM Tris–HCl pH 7.5 partitioned almost completely into the S100 cytosolic fraction (Fig. 5a), whereas integral membrane proteins LAMP1 and tyrosinase partitioned solely into the LGF and P100 fractions in both HEPES (Fig. 2) and Tris–HCl buffers (Fig. 5a and b). Similarly, in lymphoblastoid cells the small fraction of HPS protein that partitioned to the P100 fraction of cells lysed in HEPES (Fig. 2b) was abolished when cells were lysed in Tris–HCl (Fig. 5b). These results suggest that association of the HPS protein with components of the LGF in melanoma cells is dependent on a protein–membrane interaction that is subject to disruption by Tris–HCl.
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To assess directly whether the HPS protein is associated with a membrane component of the LGF via a weak interaction that is subject to disruption, the PNS fraction was prepared from FME melanoma cells lysed in cell fractionation buffer (CF; see Materials and Methods) containing 25 mM HEPES, and aliquots were then adjusted to different concentrations of Tris–HCl or NaCl and centrifuged to yield the LGF and 10 000 g cytoplasmic supernatant (S10) fractions. As shown in Figure 5c, as the concentration of Tris–HCl in the PNS buffer was increased above zero, the amount of HPS protein associated with the subsequent LGF fraction was progressively reduced and partitioning into the S10 fraction was correspondingly increased, with no effect on partitioning of the integral melanosomal protein tyrosinase to the LGF. Identical results were obtained with increasing concentrations of NaCl; partitioning of the HPS protein to the LGF fraction was reduced even by 50 mM NaCl and abolished by 500 mM NaCl, with no effect on partitioning of tyrosinase to the LGF (data not shown). These results indicate that the HPS protein in the LGF fraction of FME melanoma cells is associated with a membrane, and that this association is disrupted by Tris–HCl and NaCl under conditions in which association of integral membrane proteins remains intact.
HPS is a component of a high molecular weight complex
To determine whether the HPS protein is part of a complex, FME melanoma and normal human lymphoblastoid cells were lysed in buffer containing 25 mM HEPES, cytoplasmic fractions were prepared as above, and these were further fractionated by gel filtration through Superdex 200, followed by western blot analysis. As shown in Figure 6, the HPS protein from cytoplasmic S10 supernatant fractions from both FME melanoma (Fig. 6a) and lymphoblastoid (Fig. 6b) cells eluted as a homogeneous species with apparent molecular mass of ~200 kDa, much larger than the molecular masses of 79.3 and 75.9 kDa of the two HPS protein isoforms (Fig. 1a). The identical elution profile was obtained for HPS protein from cytoplasmic S10 fraction from FME melanoma cells lysed in buffer containing 100 mM Tris–HCl (data not shown). These results indicate that cytosolic HPS protein is part of an ~200 kDa complex that is insensitive to Tris–HCl.
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In contrast, HPS protein from FME melanoma LGF, solubilized in CHAPS–HEPES, eluted predominantly in a peak with apparent molecular mass of >500 kDa (Fig. 6c). Addition of 100 mM Tris–HCl pH 7.5 to the LGF before application to the column shifted elution of the HPS protein to ~200 kDa (Fig. 6d), the same position as the cytosolic complex (Fig. 6a). Similar results were obtained on gel filtration analyses of the 100 000 g pellet fractions (P100) from both FME melanoma and lymphoblastoid cells (data not shown). Together, these results indicate that the HPS protein in the LGF of melanoma cells is part of a >500 kDa complex, and that Tris–HCl dissociates this complex into the ~200 kDa cytosolic HPS complex, most likely by disrupting interaction with membrane components. A much smaller amount of the >500 kDa complex is also present in the P100 fraction, in both melanoma and lymphoblastoid cells.
Subcellular localization by confocal microscopy and digital fluorescence microscopy
To define further the cytoplasmic large granular/organellar compartment that contains the HPS protein in melanotic cells, we carried out confocal and digital immunofluorescence microscopy of normal human fibroblasts, normal melanocytes and FME melanoma cells, using antibodies to HPS, tyrosinase, Trp-2 (another melanosomal membrane protein), LAMP1 and a trans-Golgi network marker, TGN46.
The HPS protein was readily detected in the cytoplasm of melanocytes, FME melanoma and fibroblasts. As shown in Figure 1b, in fibroblasts, a non-melanotic cell type, the HPS protein is distributed relatively homogeneously throughout the cytoplasm. In contrast, in normal melanocytes (Fig. 7a) and in FME melanoma cells (Fig. 7b) a large fraction of the HPS protein is concentrated in the perinuclear region, with a somewhat granular pattern, superimposed on a generalized non-particulate distribution throughout the cytoplasm. Higher-resolution, digital microscopy of FME melanoma cells (Fig. 7c) showed that the perinuclear HPS protein includes a somewhat globular component that is suggestive of organelles.
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Confocal immunofluorescent imaging of double-labeled normal melanocytes and FME melanoma cells showed that at least part of the perinuclear granular component of the HPS protein appears to correspond to early-stage melanosomes. As shown in Figure 8a, in normal melanocytes tyrosinase is distributed in a granular pattern characteristic of melanosomes (panel 2), and those melanosomes located in the vicinity of the nucleus also appear to contain HPS (panels 1 and 3). Reproducibly, there was a much weaker HPS signal in melanosomes located more peripherally in the cytoplasm, in the melanocyte dendrites (Fig. 8b, panels 1–7). These results suggest that the HPS protein is specifically associated with early-stage melanosomes being formed in the perinuclear region, but not with later-stage melanosomes that have moved out into the cytoplasmic dendrites. This conclusion was supported by double-labeling of FME melanoma cells, which contain pre- and early-stage melanosomes but few late-stage melanosomes; most of the tyrosinase signal was concentrated in perinuclear granules, most of which were also labeled by HPS (Fig. 8b, panels 8 and 9). Rare peripheral melanosomes showed little HPS staining. Identical results were obtained by double-labeling melanocytes and melanoma cells for HPS and the melanosomal marker Trp-2 (data not shown).
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In contrast, there was little apparent concordance in the distributions of HPS and the lysosomal marker LAMP1. As shown in Figure 8a, although the HPS (panel 4) and LAMP (panel 5) signals in normal melanocytes appear superficially similar, double-labeling (panel 6) shows substantial differences, with no definitive co-localization of signals. Identical results were obtained for FME melanocytes and for normal fibroblasts (data not shown). These results are consistent with our cell fractionation data, which indicated that the HPS protein is not a component of lysosomes (Fig. 2b). Furthermore, there was no similarity at all between the distribution of HPS protein and that of TGN46, a marker for the TGN (data not shown). Together, these results indicate that the large granular/organellar component of HPS protein in melanocytes and melanoma cells is located principally in the perinuclear region of the cytoplasm, in a compartment that appears to include early-stage melanosomes but probably not lysosomes.
Immunoelectron microscopy
To define more precisely the cytoplasmic large granular/organellar compartment that contains the HPS protein, we carried out immunoelectron microscopy of FME and MNT1 melanoma cells and human lymphoblastoid cells using HPS mAb hHPS5 and immunogold labeling. To reduce generalized cytoplasmic labeling due to cytosolic HPS, fixation conditions were used that would not retain cytosolic material, resulting in low but quite specific and reproducible immunogold labeling. In FME melanoma cells (Fig. 9a), which contain early-stage melanosomes but no later-stage melanosomes, gold particles labeled the periphery of tubulovesicular structures and small, non-coated vesicles in the vicinity of the Golgi apparatus, and the membrane surrounding very early-stage melanosomes; coated vesicles were not labeled. In MNT1 melanoma cells (Fig. 9b), which contain melanosomes of all stages, gold particles again principally labeled the periphery of tubulovesicular structures and small non-coated vesicles located near the Golgi apparatus, as well as the membrane surrounding early-stage melanosomes, some of which appeared to contain labeled vesicles. In lymphoblastoid cells, immunogold particles labeled only peri-Golgi tubulovesicular structures and non-coated vesicles (data not shown). These results thus indicate that the non-cytosolic component of HPS protein is associated with tubulovesicular structures and small, non-coated vesicles in the peri-Golgi region and, in melanotic cells, with early-stage melanosomes.
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DISCUSSION |
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In melanocytes, defective pigmentation in HPS results from decreased and aberrant melanosomes (19,20). In platelets, tendency to bleeding results from defective platelet aggregation due to decreased numbers of platelet dense granules, which are necessary for formation of primary clots. Also, lysosomes of many cell types, in particular of the reticuloendothelial system, appear distended and tend to accumulate autofluorescent material that has some characteristics of ceroid (1,2). Furthermore, mice with the homologous defect, pale-ear (9,21), exhibit abnormalities of lysosomal enzyme trafficking (22,23) and immunological defects thought to result from defective lysosomal or granular function (2). HPS has thus been thought to result from defects of a protein required for the biogenesis, structure or function of these various membrane-bound organelles, at least in those cell types that are involved in the pathology of the disorder.
The function of the HPS protein has been enigmatic, as the HPS polypeptide sequence is not homologous to any other known proteins and contains no peptide motifs that might provide clues to its function (5). Comparison of the human, mouse and rat HPS amino acid sequences has highlighted no obviously conserved domains that might be of particular functional importance (9; J. Oh and R.A. Spritz, unpublished data), and there is no apparent homolog to the HPS protein in yeast or worms. Also, no missense mutations have yet been found in human HPS patients that might indicate regions critical for normal function.
We have begun to address the function of the HPS protein by defining its subcellular location and its characteristics in cultured human cells. We find that the HPS protein is neither N-glycosylated nor an integral membrane protein, but that it is a component of two distinct high molecular weight complexes, a >500 kDa complex that is membrane associated and an ~200 kDa complex that is cytosolic. In melanotic cells the HPS protein is approximately evenly divided between the >500 kDa and ~200 kDa complexes, whereas in non-melanotic cells almost all of the HPS protein is in the ~200 kDa cytosolic complex. The >500 kDa membrane-associated HPS complex of melanotic cells is localized principally in the perinuclear region, in association with tubulovesicular and non-coated vesicular structures in the region of the Golgi apparatus, and with the peripheral membrane of nascent and early-stage melanosomes. The much smaller amount of >500 kDa membrane-associated HPS complex found in lymphoblastoid cells is likewise associated with peri-Golgi tubulovesicular structures and small, non-coated vesicles.
What is the relationship between the ~200 and >500 kDa HPS complexes? Even low concentrations of Tris–HCl or NaCl convert the >500 kDa complex to a species similar or equivalent to the ~200 kDa cytosolic complex, apparently by dissociating membrane components. The ~200 kDa cytosolic HPS complex is insensitive to Tris–HCl and NaCl, and might be composed of HPS in association with other proteins or it might represent a multimer of the HPS protein itself. One attractive hypothesis is that the ~200 kDa complex is a cytosolic reservoir of HPS protein that becomes associated with small, non-coated peri-Golgi vesicles involved in trafficking certain ‘cargo proteins’ to specific organelles, including nascent and early-stage melanosomes, during their early biogenesis. During subsequent stages of organellogenesis the membrane-associated HPS complex might then dissociate and either be recycled to the cytosolic reservoir or be degraded.
In fact, the pathway of melanosomal biogenesis is not well understood. It has been suggested that the earliest elements of the melanosomal series (‘pre-melanosomes’) might bud from the endoplasmic reticulum, subsequently fusing with Golgi-derived vesicles that contain the specific proteins required for melanin synthesis, such as tyrosinase and Trp-2, to form early-stage melanosomes (24). Recent attention has focused on the AP-3 adaptin-dependent pathway of trafficking newly synthesized cargo proteins from the TGN into transport vesicles, for eventual delivery to a subset of nascent organelles (3,4,25,26). In the mouse, ~14 different genes, including Hps/pale-ear, are associated with HPS-like mutant phenotypes that involve abnormalities of melanosomes, lysosomes and/or platelet granules (3,4,22,23). Two of these mouse loci, mocha (27) and pearl (28), encode subunits of the AP-3 complex itself, and a variant form of human HPS has likewise been shown to result from mutations in the gene encoding the AP-3 ß3A subunit (29). Recently, the gene for another HPS-like mouse mutant, pallid, has been shown to encode a novel syntaxin 13-interacting protein that seems likely to be involved in fusion of vesicles to early endosomes (30), a later stage of early organellogenesis than those thought to involve AP-3. The HPS protein is clearly not a component of the AP-3 complex, and the HPS and AP-3 complexes do not co-immunoprecipitate (J. Oh and R.A. Spritz, unpublished data), suggesting that they may not interact directly. Instead, our current data suggest that the HPS complex may participate in the biogenesis of nascent organelles in a novel way, involving trafficking of cargo proteins via non-coated vesicles that derive from tubulovesicular precursors in the region adjacent to the Golgi apparatus.
The evident involvement of the HPS protein in melanosomal biogenesis is expected, given the pigmentary defects and abnormalities of the melanosome found in patients with the disorder (1,19,20) and in pale-ear mutant mice (31). However, our finding that the HPS protein is apparently not a component of lysosomes is unexpected, as patients with HPS exhibit cellular inclusions and accumulations of autofluorescent material thought to reflect lysosomal dysfunction (1,3,4,19), and pale-ear mice exhibit defective secretion of lysosomal enzymes by the kidney (32). It may be that the HPS complex is involved in lysosomal biogenesis, but that its association with nascent lysosomes is earlier or more transient than with early melanosomes. Or it may be that HPS dysfunction impacts the lysosome only indirectly, perhaps accounting for the very mild lysosomal defects in patients with HPS compared with patients with classical lysosomal storage disorders. The biogenetic relationship between the melanosome and the lysosome has long been a mystery, and it is clear that much more work will be required to finally elucidate the connection between these two organelles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell cultures
Normal human melanocytes were cultured in melanocyte growth medium (MGM; Clonetics, San Diego, CA). FME (33) and MNT1 (34) human melanoma cells were cultured in Ham’s F10 with 1% penicillin–streptavidin and 10% fetal bovine serum (FBS). Fibroblasts from clinically normal (GM04930) and HPS-affected (GM14609) individuals were obtained from the Coriell Cell Repository (Camden, NJ) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 1% penicillin–streptavidin and 15% FBS. Lymphoblastoid cells from normal or HPS individuals (GM14609) were cultured in RPMI1640 with 1% penicillin–streptamycin and 15% FBS.
Antibodies
Human HPS cDNA segments encoding amino acids 1–463 and 462–700 were cloned individually in pET28a(+), the resultant plasmids were introduced into Escherichia coli BL21(DE3)pLysS, and expression of the HPS protein segments was induced with IPTG. The two HPS protein segments were purified by 7.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE), eluted, and a 1:1 mixture of the two was used to immunize rabbits and mice to raise polyclonal antisera and mAbs, respectively. Rabbit polyclonal antisera were affinity-purified using the pooled HPS protein segments. mAbs were used as tissue culture supernatants. Additional antibodies used included human LAMP1 mAb (H4A3; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse tyrosinase rabbit polyclonal antisera [
PEP7 (35)], mouse Trp-2 rabbit polyclonal antisera [EP8 (35)], human tyrosinase rabbit polyclonal antisera [PEP7h (36)], human TGN46 rabbit polyclonal antisera (37) and human Mre11 rabbit polyclonal antisera (38).
Cell fractionation analyses
FME melanoma, lymphoblastoid and fibroblast cells (107–108) were harvested by centrifugation, washed twice with cold phosphate-buffered saline (PBS), and were resuspended in either CF–HEPES lysis buffer (0.25 M sucrose, 25 mM HEPES–KOH pH 6.8, 25 mM KCl, 2.5 mM MgCl2) or CF–Tris lysis buffer (0.25 M sucrose, 50 mM Tris–HCl pH 7.5, 25 mM KCl, 5 mM MgCl2). All solutions contained 5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM benzamidine and 20 µg/ml aprotinin as proteolytic inhibitors. After incubation on ice for 10 min, cells were homogenized on ice with 100 strokes of a precooled glass Dounce homogenizer (B pestle); cell lysis was monitored by uptake of trypan blue dye. Cell homogenates were centrifuged at 1000 g for 10 min at 4°C. Pellets were resuspended in the same lysis buffer containing 1.6 M sucrose, homogenized with 5–10 strokes and centrifuged through the same lysis buffer containing 2.1 M sucrose at 100 000 g for 1 h at 4°C to isolate cytoplasm-free nuclei (Nu). The supernatant (PNS) was then centrifuged at 10 000 g for 10 min at 4°C to obtain a large granule fraction (LGF; pellet) and supernatant (S10). The S10 supernatant was centrifuged at 100 000 g for 1 h at 4°C to yield pellet (P100) and supernatant (S100) fractions (39). The Nu, LGF and P100 pellets were resuspended in the starting lysis buffer, normalized for cell number, and equal volumes of the pellet and supernatant extracts were separated by 7.5% SDS–PAGE along with prestained molecular size standards and were transferred electrophoretically to nitrocellulose membranes for western blot analysis.
Western blot analyses
Membranes were blocked by incubation at room temperature for 1 h in 100 mM Tris–HCl pH 7.5, 0.9% NaCl, 0.1% Tween-20 (TTBS) containing 5% bovine serum albumin (BSA), and were then incubated for 1 h at room temperature with primary antibody in the same solution. After four washes in TTBS, bound antibodies were detected with goat anti-mouse or goat anti-rabbit horse radish peroxidase (HRP) conjugates (Pierce, Rockford, IL) at 1:20 000 dilutions, as appropriate, and the SuperSignal Substrate Western Blotting kit (Pierce). Primary antibodies and corresponding dilutions were affinity-purified HPS polyclonal antisera hHPS504 at 1:40; HPS mAb hHPS5 at 1:500; LAMP1 mAb H4A3 at 1:500; mouse tyrosinase polyclonal antisera PEP7 at 1:400; and human tyrosinase polyclonal antisera PEP7h at 1:500. To remove the bound antibody and immunodetection system, filters were incubated with 2% w/v SDS, 62.5 mM Tris–HCl pH 6.8, 100 mM ß-mercaptoethanol for 15 min at 70°C, and were then washed twice with TTBS for 10 min each at room temperature.
N-glycosylation analyses
For endoglycosidase H treatment, 5 x 106 FME melanoma cells were lysed following the manufacturer’s recommendations, and the cell lysate was incubated with 0 or 5 U/µl endoglycosidase H (New England Biolabs, Beverly, MA) and assayed by western blotting as above. For assay of tunicamycin inhibition of N-glycosylation, 1.5 x 106 FME melanoma cells were cultured in 0, 0.2 or 1.0 µg/ml tunicamycin (Sigma, St Louis, MO) for 5 days, lysed, and proteins were analyzed by western blotting.
Transmembrane analyses
For Triton X-114 fractionation (17), LGF and S10 fractions were prepared from 108 FME melanoma or lymphoblastoid cells as above. The LGF was solubilized with 1% Triton X-114 containing 10 mM Tris–HCl pH 7.5, 0.15 M NaCl, 40 µg/ml aprotinin, 10 mM benzamidine, and the supernatant was adjusted to 1% Triton X-114. Both were then warmed at 37°C for 5 min and centrifuged at 700 g for 5 min to separate aqueous and detergent phases. Fractions were normalized for cell number and assayed by western blot analysis as above.
For assay of membrane protein stripping by Tris–HCl and NaCl, FME melanoma cells were suspended in CF–HEPES and the PNS fraction prepared as above. Aliquots of PNS were then adjusted to 0, 10, 25, 50, 75 or 100 mM Tris–HCl pH 7.5 or 0, 50, 100, 200, 300 or 500 mM NaCl, incubated on ice for 10 min, and centrifuged at 10 000 g for 10 min to yield LGF pellet and supernatant (S10) fractions. LGF pellets were resuspended in CF–HEPES in the starting volume, and equal volumes of LGF and S10 were assayed by 7.5% SDS–PAGE and western blot analysis as above.
Gel filtration analyses
FME melanoma and normal human lymphoblastoid cells (2 x 108) were harvested by centrifugation, washed twice with cold PBS, and resuspended in 1 ml of CF–HEPES. LGF and P100 pellets were prepared as above and were resuspended in either 0.5 ml of CHAPS–HEPES buffer (0.25 M sucrose, 25 mM HEPES–KOH pH 6.8, 25 mM KCl, 2.5 mM MgCl2, and 10 mM CHAPS) or CHAPS–Tris buffer (0.25 M sucrose, 25 mM HEPES–KOH pH 6.8, 25 mM KCl, 2.5 mM MgCl2, 5 mM CHAPS and 100 mM Tris–HCl pH 7.5), incubated on ice for 1 h and then centrifuged at 10 000 g for 10 min to remove insoluble material. The cytosolic fraction (S100) was prepared in 1 ml of either CF–HEPES or CF–Tris as above. Fractions of 0.5 ml were loaded onto a Superdex 200 prep grade column (0.7 x 50 cm FLEX; Kimble/Kontes, Vineland, NJ) equilibrated and eluted at 4°C with either CF–HEPES or CF–HEPES plus 100 mM Tris–HCl pH 7.5, as appropriate, at a flow rate of 0.2 ml/min. Column calibration was carried out using ferritin (468 kDa), catalase (204 kDa) and BSA (67 kDa) as size standards. Column fractions (1.0 ml) were collected and aliquots of the fractions were assayed by SDS–PAGE and western blot analysis as above. All analyses were repeated at least two times.
Confocal and digital immunofluorescence microscopy
Normal human fibroblasts, melanocytes and FME melanoma cells were cultured for 12–24 h on glass coverslips coated with polylysine. Cells were washed with PBS, fixed for 10 min in methanol at –20°C, washed three times with PBS, permeabilized in 50 mM NaCl, 3 mM MgCl2, 200 mM sucrose, 10 mM HEPES pH 7.9, 0.1–0.3% Triton X-100 for 5 min at room temperature, and washed twice with PBS. Non-specific antibody binding was blocked for 30 min at room temperature in PBS containing 5% normal goat serum (NGS), followed by three washes with PBS. Coverslips were incubated sequentially for 1 h each at room temperature with first (HPS mAb hHPS5 at 1:500 dilution or human LAMP1 mAb H4A3 at 1:500) and second (human LAMP1 rabbit polyclonal antisera-FITC at 1:25, mouse tyrosinase rabbit polyclonal antisera PEP7h at 1:50, mouse Trp-2 rabbit polyclonal antisera PEP8 at 1:50 or human TGN46 rabbit polyclonal antisera at 1:500) primary antibodies in 1% NGS in PBS, with three washes in PBS after each incubation. Coverslips were then incubated for 1 h at room temperature in 1% NGS in PBS containing secondary antibody (either goat anti-mouse IgG-rhodamine at 1:100 or sheep anti-mouse IgG-Cy3 at 1:2000 plus goat anti-rabbit IgG-FITC at 1:40, or goat anti-rabbit IgG-rhodamine at 1:100 dilution when the first primary antibody was -LAMP1-FITC). Coverslips were then washed three times in PBS and mounted on glass in antifade mounting medium containing 2.3% 1,4-diazabiocyclo[2,2,2]octane (Sigma), 90% glycerol, 0.1 M Tris–HCl pH 8.0 and sealed with nail polish. For confocal microscopy, samples were viewed under epifluorescence using a Bio-Rad (Hercules, CA) MRC 1000 scanhead mounted transversely to an inverted Nikon (Tokyo, Japan) Diaphot 200 microscope with x100 PlanApo objective, triple dichroic filter set and 15 mM krypton/argon mixed gas air-cooled laser controlled by Bio-Rad MRC-1024 Laser Sharp (v2.1T) software. For digital microscopy, samples were viewed under epifluorescence using a Nikon Diaphot TMD microscope with x100 PlanApo objective, Xenon lamp and filter wheels, fluorescent filters, cooled CCD camera and stepper motor. Images were collected, merged, deconvolved and re-normalized using Slidebook software (Intelligent Imaging Innovations, Denver, CO).
Electron microscopy
Human lymphoblastoid cells and FME and MNT1 melanoma cells were fixed either with 2% paraformaldehyde in 0.2 M phosphate buffer pH 7.4 (PB) or with 2% paraformaldehyde, 0.125% glutaraldehyde for 2 h at room temperature. Fixed cells were processed for ultrathin sectioning and immunolabeling (40) by washing with PB and PB-glycine (50 mM) and embedding in 7.5% gelatine. Small blocks were infiltrated with 2.3 M sucrose at 4°C for 2 h and frozen in liquid N2. Ultrathin cryosections prepared with a Leica (Wetzlar, Germany) ultracut FCS were retrieved with 1% methylcellulose, 1.15 M sucrose. Sections were put on formvar-coated grids, thawed and labeled with HPS mAb hHPS5, followed by rabbit anti-mouse IgG (Dako, Carpinteria, CA) and protein A–gold (Dr J.W. Slot, Department of Cell Biology, Utrecht University, The Netherlands). Sections were contrasted and embedded in a mixture of methylcellulose and uranyl acetate and viewed under a CM120 Twin Phillips electron microscope.
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ACKNOWLEDGEMENTS |
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We thank Drs Richard Burgess, John Petrini, Kathryn Howell, Alex Franzusoff and Margaret Neville for invaluable advice and assistance, Vincent J. Hearing Jr for
PEP7, PEP7h and PEP8 antisera, Douglas M. Fambrough for LAMP1 antisera, George Banting for TGN46 antisera, John Petrini for Mre11 antisera, and Brian Bordini for technical assistance. This work was supported by Clinical Research grant 6-0281 from the March of Dimes Birth Defects Foundation and grant AR39892 from the National Institutes of Health.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ Present address: Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA
¶ Present address: Cereon Genomics, LLC, Cambridge, MA 02139, USA
To whom correspondence should be addressed. Tel: +1 303 315 0409; Fax: +1 303 315 0407; Email: richard.spritz@uchsc.edu
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REFERENCES |
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