From the
Human Medical Genetics Program and ¶Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 80262 and the
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
Received for publication, January 6, 2003 , and in revised form, March 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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Most human HPS patients have mutations of either HPS1 or HPS4. Human HPS1 is a 700-amino acid, 79.3-kDa, non-membrane protein, and HPS4 is a 708-amino acid, 76.9-kDa, non-membrane protein, both unrelated to other known proteins and containing no sequence motifs that might provide clues to their functions in organellar biogenesis. Nevertheless, several lines of evidence suggest that the HPS1 and HPS4 proteins may be functionally related. First, Hps1-mutant (pale-ear; ep) and Hps4-mutant (light-ear; le) mice exhibit very similar phenotypes, grossly and at the biochemical and cellular ultrastructural levels (19, 20, 21). Second, ep/ep, le/le double-homozygous mutant mice have a phenotype that is the same as that of the two homozygous single mutants (22). Third, the HPS1 protein is absent in cells of both Hps1-mutant (ep) and Hps4-mutant (le) mice (7). Finally, the HPS1 and HPS4 proteins partially co-localize in cytoplasmic vesicles of transfected melanoma cells (7). Together, these data strongly suggest a functional relationship or interaction between the HPS1 and HPS4 proteins.
Herein, we show that the human HPS1 and human HPS4 proteins are both components of a 500 kDa complex associated with vesicles and organelles that we named biogenesis of lysosome-related organelles complex 3 (BLOC-3). Within BLOC-3, HPS1 and HPS4 are contained in a discrete
200 kDa module that we term BLOC-4. It is likely that BLOC-4, and certainly BLOC-3, contain additional protein components not yet identified. Our findings indicate that, as components of BLOC-3 and BLOC-4, the HPS1 and HPS4 proteins play a coordinated role in trafficking of proteins to newly synthesized organelles.
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EXPERIMENTAL PROCEDURES |
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Recombinant Protein Expression, Purification, and Antibody ProductionpDEST15-HPS4 was introduced into E. coli BL21-CodonPlus-RP (Stratagene), and GST-HPS4 fusion protein was purified from inclusion bodies (U.S. Dept. Commerce/NOAA/NMFS/NWFSC/Molecular Biology Protocols; URL: micro.nwfsc.noaa.gov/protocols/inclusion.html), and used as immunogen to prepare anti-hHPS4 polyclonal antisera in rabbits and HPS4 monoclonal antibodies in mice (Covance). Monoclonal antibodies to HPS1 were described previously (23). Monoclonal antibody to His tag was from Clontech.
Derivation and Transfection of ep- and le-Mutant Mouse MelanocytesThe wild-type mouse melanocyte line, melan-a (24) and melan-ep and melan-le melanocytes (7) have been described previously. Cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 200 nM phorbol 12-myristate 13-acetate at 37 °C in a 10% CO2 atmosphere. Melan-ep or melan-le melanocytes were transfected with the pIREShyg2-HPS1 and pIREShyg2-HPS4 constructs using FuGENE 6 Transfection Reagent (Roche Applied Science), and stable transformants were selected in culture media containing 300 µg/ml hygromycin B. Human MNT1 melanoma cells (25) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% heat-inactivated fetal bovine serum, 10% Panserin 401 (PAN Biotech), 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 100 nM Na-pyruvate at 37 °C in a 5% CO2 atmosphere. Cells were visualized by standard bright-field and Hoffman illumination microscopy.
ImmunoblottingProteins were separated in 7.5% SDS-polyacrylamide and transferred to Immobilon-P membranes (Millipore). Membranes were blocked in 5% fat-free milk in phosphate-buffered saline for 1 h at room temperature, and then were incubated in phosphate-buffered saline containing 3% bovine serum albumin, 0.2% Tween 20, and the appropriate primary antibody for 1 h at room temperature. Membranes were then washed extensively, incubated with appropriate horseradish peroxidase-conjugated secondary antibody (Pierce) for 1 h at room temperature, and washed again. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Blots were calibrated with pre-stained molecular weight standards (Invitrogen). For analysis of HPS4·HPS1 complexes, immunoblots of gel filtration fractions, previously probed with anti-HPS1 antibody (23), were stripped in 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.7, for 30 min at 50 °C and were reprobed as described above.
Reverse Transcription-PCRRNA from melan-a and melan-le melanocytes was isolated using the TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using SuperScript II RNase H- reverse transcriptase and oligo(dT)25 as primer (Invitrogen). PCR was performed using equivalent amounts of melan-a and melan-le RNAs, Platinum Taq DNA polymerase (Invitrogen), and mouse Hps1 forward (5'-GACTTCCGCCTGCTCCTACTTCCT-3') and reverse (5'-GCGTGAGCAGCTCATAGCAC-3') primers derived from exons 20 and 22, respectively.
Co-immunoprecipitation AssaysMNT1 cells were lysed in 50 mM Na-phosphate buffer, pH 8.0, 300 mM NaCl, 1% Tween 20, and protease inhibitors (Roche Applied Science). 600 µl of fresh lysate was incubated with 40 µl of rabbit anti-HPS4 antisera, 40 µl of HPS1 monoclonal antibody, 40 µl of rabbit preimmune immunoglobulins (Santa Cruz Biotechnology), or 40 µl of mouse preimmune immunoglobulins (Santa Cruz Biotechnology) for 1 h at 4 °C with gentle agitation. Protein G-Sepharose 4 Fast Flow (Amersham Biosciences), pre-washed in lysis buffer, was added and the samples were incubated for 1 h at 4 °C. Immunoprecipitates were washed several times in lysis buffer and then boiled for 5 min in Laemmli sample buffer.
To test the effect of Tris-HCl on the BLOC-3 complex, we purified the vesicular-organellar fraction from MNT1 cells as described (23) using 50 mM phosphate buffer, pH 8.0, as above. The fraction was then resuspended in either phosphate buffer or Tris buffer (0.25 M sucrose, 100 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, 1% Tween 20), immunoprecipitated using HPS1 antibody or HPS4 antibody, and analyzed by Western blot analysis using HPS4 antibody or HPS1 antibody, respectively.
For purification of His-tagged proteins, cells were lysed in phosphate-buffered saline containing 1% Nonidet P-40 and protease inhibitors, and lysates were incubated with TALON metal affinity resins (Clontech) at room temperature for 30 min, washed several times in lysis buffer, and boiled for 5 min in Laemmli sample buffer.
Yeast Two-Hybrid AssaysFull-length mouse Hps1 and Hps4 cDNAs were inserted in-frame in the DNA binding domain (pGBKT7) and activation domain (pGADT7) Gal4 transcription factor vectors of the Matchmaker Gal4 two-hybrid system 3 (Clontech), and verified by sequencing. Fusion constructs were co-transformed into Saccharomyces cerevisiae strain AH109. At least two colonies of each double transformant growing on low-stringency medium (-Trp/-Leu) were suspended in low-stringency broth and grown at 30 °C for 16 h with shaking. Cell cultures were adjusted to 0.4 OD at 600 nm, and 3 µl from each co-transformation were spotted onto low- and high-stringency (-Trp/-Leu/-His/-Ade) SD (Clontech) plates containing X- Gal. Plates were incubated at 30 °C for 5 days and monitored for growth and blue color by visual inspection.
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RESULTS |
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We showed previously that the HPS1 protein is unexpectedly absent in tissues from Hps4-null (le-mutant) mice as well as from Hps1-null (ep-mutant) mice (7). As shown in Fig. 2, we obtained the same result on Western blot analysis of cultured melan-ep and melan-le melanocytes; both lack detectable HPS1 protein. We could not assay HPS4 protein in these cells, because our human HPS4 polyclonal antisera and monoclonal antibodies do not recognize the mouse HPS4 protein. Semiquantitative reverse transcription-PCR showed that the amount of Hps1 mRNA is approximately equal in wild-type melan-a versus melan-le cells (data not shown), indicating that HPS4 protein apparently is not required for transcription and biogenesis of Hps1 mRNA. These results suggest instead either that HPS4 is required for biosynthesis of HPS1 or that absence of HPS4 destabilizes HPS1.
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To determine whether HPS4 is required for biosynthesis of HPS1, we overexpressed human HPS4 in Hps1-null melan-ep mouse melanocytes by stable transformation with a human HPS4 cDNA expression plasmid and human HPS1 in Hps4-null melan-le melanocytes by transformation with a human HPS1 expression plasmid. As expected, melan-ep cells transfected with the HPS1 expression plasmid produced abundant human HPS1 protein (Fig. 2, lane 4) and were complemented to a normal pigmentation and melanosomal cellular phenotype (Fig. 1). Likewise, melan-le cells transfected with the HPS4 expression plasmid produced abundant human HPS4 protein (Fig. 2, lane 8) and were complemented to a normal cellular phenotype (Fig. 1). Moreover, over-expression of human HPS4 protein in transfected melan-le cells fully restored expression of endogenous HPS1 protein (Fig. 2, lane 8). In contrast, melan-ep cells transfected with the HPS4 expression plasmid produced abundant human HPS4 protein (Fig. 2, lane 5), although they of course still expressed no endogenous HPS1 protein (Fig. 2) and showed no apparent correction of the abnormal pigmentary or melanosomal phenotype (Fig. 1). However, melan-le cells transfected with the HPS1 expression plasmid produced abundant human HPS1 protein (Fig. 2, lane 7), demonstrating that HPS4 is not required for biosynthesis of HPS1. Interestingly, these cells showed partial correction of the abnormal cellular phenotype (Fig. 1) presumably because of correction of the secondary HPS1 deficit in these cells. These results show clearly that HPS4 is not required for biosynthesis of HPS1 protein but that HPS4 instead most likely stabilizes HPS1 as part of a protein complex.
HPS1 and HPS4 Co-immunoprecipitateTo determine whether HPS1 and HPS4 are in a protein complex, we independently immunoprecipitated HPS1 and HPS4 from human MNT1 melanoma cells and subsequently assayed HPS4 protein in the HPS1 immunoprecipitate and vice versa. As shown in Fig. 3, monoclonal antibody to human HPS1 detected the typical HPS1 doublet in the HPS4 immunoprecipitate. Likewise, polyclonal antibody to human HPS4 detected a single band in the HPS1 immunoprecipitate corresponding to human HPS4. Thus, HPS1 and HPS4 indeed co-immunoprecipitate, indicating that they are in a protein complex. Similarly, we detected interaction between the human HPS4 and mouse HPS1 proteins in vivo, assayed both by co-immunoprecipitation of mHPS1-hHPS4 from melan-le cells transfected by the pIREShyg2-HPS4 expression plasmid (not shown) and by expressing human His-tagged HPS4 in transfected mouse melan-le melanocytes, purifying the His-tagged HPS4 protein on a metal ion column and immunoblotting the bound protein for HPS1 (Fig. 3). Together, these results demonstrate that HPS1 and HPS4 are components of a protein complex in vivo.
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HPS1 and HPS4 Do Not Interact in a Yeast Two-hybrid System, but HPS4 Binds to ItselfTo determine whether HPS1 and HPS4 interact directly, we used a yeast two-hybrid system to test interaction between full-length HPS1 and HPS4. Appropriate combinations of mouse HPS1 and HPS4 binding domain and activation domain constructs were co-transfected into yeast strain AH109 and the co-transformants were plated on SD/-Ade/-His/-Leu/-Trp. As shown in Fig. 4, we saw no growth for any of the HPS1·HPS4 co-transformants in any combination, indicating that mouse HPS1 and HPS4 do not interact directly, at least in this yeast system. However, as shown in Fig. 4, we observed vigorous growth and blue color for the HPS4·HPS4 combination, indicating that HPS4 monomers interact strongly with each other. We observed no apparent HPS1·HPS1 interaction. We obtained identical results using human HPS1 and HPS4 constructs (not shown). These results indicate that HPS1 and HPS4 may not interact directly but that HPS4 self-associates to at least a dimer.
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HPS1 and HPS4 Are Components of a 500-kDa Cytoplasmic Vesicular-Organellar Complex, BLOC-3We showed previously that HPS1 is part of a >500-kDa cytoplasmic vesicular-organellar complex and a
200-kDa cytosolic complex (23). To determine which of these high molecular mass HPS1 complexes contains HPS4, we reinterrogated the immunoblots used in the previous study, now using antisera to HPS4. As shown in Fig. 5A, HPS1 and HPS4 are both contained in the same >500-kDa cytoplasmic vesicular/organellar fraction from FME melanoma cells. In contrast, the HPS4 protein was undetectable in cytosolic fractions from both FME melanoma cells (Fig. 5B) and human lymphoblastoid cells (not shown). These results demonstrate that the >500-kDa cytoplasmic vesicular-organellar complex represents BLOC-3.
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A Discrete 200-kDa HPS1·HPS4 Complex, BLOC-4, Is a Module of BLOC-3We showed previously that treatment of the >500-kDa BLOC-3 complex with 100 mM Tris-HCl results in release of HPS1 as a
200-kDa post-Tris HPS1 complex, which we speculated might be identical to the
200-kDa HPS1 complex observed in the cytosol (23). To determine whether HPS4 is also released from BLOC-3 by Tris-HCl, we reinterrogated the immunoblots used in the previous study, now using antisera to HPS4. As shown in Fig. 5C, treatment of the FME melanoma vesicular/organellar fraction with 100 mM Tris-HCl results in release of both HPS1 and HPS4 into the same
200-kDa sub-fractions. Thus, HPS1 and HPS4 are both released from BLOC-3 into a
200-kDa post-Tris complex.
To determine whether HPS1 and HPS4 released from BLOC-3 by Tris-HCl reside together in a single 200-kDa post-Tris complex or in two different
200-kDa post-Tris complexes, we tested whether HPS1 and HPS4 could still be coimmunoprecipitated after dissociation from BLOC-3 by 100 mM Tris-HCl. As shown in Fig. 6, treatment of the vesicular/organellar fraction of MNT1 melanoma cells with Tris-HCl did not affect subsequent co-immunoprecipitation of HPS1 and HPS4, indicating that these two proteins are released from BLOC-3 in a single
200-kDa post-Tris complex that we designate BLOC-4. BLOC-4 thus represents a discrete module of BLOC-3 and is clearly distinct from the
200 kDa cytosolic HPS1 complex, which does not contain HPS4 (Fig. 5B) and which we tentatively designate BLOC-5. The fractionation scheme and relationship of BLOC-3, BLOC-4, and BLOC-5 are shown in Fig. 7.
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DISCUSSION |
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There has been considerable progress recently in understanding the relationships among these HPS-related genes, and several of the corresponding proteins have now been shown to be associated with each other in poly-protein complexes. Mouse mocha (mh)/human AP3D1 and mouse pearl (pe)/human AP3B1 both encode components of the heterotetrameric AP-3 adaptor complex (27). Mouse pallid (pa), muted (mu), and cappuccino (cno) encode components of a novel biogenesis of lysosome-related organelles complex, BLOC-1 (28, 29). Very recently, we showed that mouse ruby-eye (ru)/human HPS6 and mouse ruby-eye-2 (ru2)/human HPS5 encode components of another novel complex, BLOC-2 (8). Here, we show that the HPS1 and HPS4 proteins are components of two additional lysosome-related organellar protein complexes, BLOC-3 and BLOC-4 (Fig. 7), and that cytosolic HPS1 seems to be part of yet another complex, BLOC-5.
Absence of HPS4 seems to result in destabilization of HPS1. Similar observations have been made for other HPS-related protein complexes. Steady-state levels of the AP-3 3A protein are decreased in mocha (Ap3d1-mutant) as well as in pearl (Ap3b1-mutant) mice (15, 30). Similarly, steady-state levels of the pallidin protein are reduced in both muted and reduced pigmentation (rp)-mutant mice (28). We have shown that the HPS1 protein is absent in light-ear (le) as well as pale-ear (ep)-mutant mice (7). However, the level of HPS1 protein is not reduced in pearl (pe; Ap3b1-mutant) mice and the levels of the four AP-3 component proteins are not reduced in pale-ear (ep; Hps1-mutant) mice (7), suggesting that HPS1 is not integrally associated with the AP-3 adaptor complex.
BLOC-4 seems to be a discrete HPS1·HPS4-containing module of BLOC-3 that is released by treatment with Tris-HCl. Studies using a yeast two-hybrid system indicate that HPS1 may not interact directly with HPS4, nor with itself, whereas HPS4 does interact with other molecules of HPS4. The relationship between BLOC-3 and BLOC-4, their other components, and the stoichiometry of these components remain to be determined. In the cytosol, HPS1 is contained in another 200-kDa complex (23) that does not contain HPS4 and which thus seems to be distinct from BLOC-4. We tentatively designate this cytosolic HPS1 complex BLOC-5.
BLOC-3 is detected in a partially purified cytoplasmic fraction that contains vesicles and organelles. We have previously shown that non-cytosolic HPS1 is principally localized in uncoated small vesicles, vesiculo-tubular structures, and early-stage melanosomes (23) and that HPS4 partially colocalizes with HPS1 in vesicles and organelles (7). BLOC-3 and its component BLOC-4 thus seem to play a key role in trafficking newly synthesized cargo proteins to nascent lysosome-related cytoplasmic organelles.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Human Medical Genetics Program, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Box B161, Denver, CO 80262. Tel.: 303-315-0409; Fax: 303-315-0407; E-mail: richard.spritz{at}uchsc.edu.
1 The abbreviations used are: HPS, Hermansky-Pudlak syndrome; BLOC, biogenesis of lysosome-related organelles complex; GST, glutathione S-transferase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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