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Hermansky–Pudlak syndrome
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The delivery of endocytosed cargo to lysosomes occurs through kissing and direct fusion of late endosomes/MVBs (multivesicular bodies) and lysosomes. Live-cell and electron microscopy experiments together with cell-free assays have allowed us to describe the characteristics of the delivery process and determine the core protein machinery required for fusion. The ESCRT (endosomal sorting complex required for transport) machinery is required for MVB biogenesis. The HOPS (homotypic fusion and vacuole protein sorting) complex is required for endosome–lysosome tethering and a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex including the R-SNARE VAMP7 (vesicle-associated membrane protein 7) mediates endosome–lysosome membrane fusion. Protein-binding partners of VAMP7 including the clathrin adaptors AP-3 (adaptor protein 3) and Hrb (HIV Rev-binding protein) are required for its correct intracellular localization and function. Overall, co-ordination of the activities of ESCRT, HOPS and SNARE complexes are required for efficient delivery of endocytosed macromolecules to lysosomes. Endosome–lysosome fusion results in a hybrid organelle from which lysosomes are re-formed. Defects in fusion and/or lysosome reformation occur in a number of lysosome storage diseases.
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Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease affecting vesicle trafficking among lysosome-related organelles. The Hps3, Hps5, and Hps6 genes are mutated in the cocoa, ruby-eye-2, and ruby-eye mouse pigment mutants, respectively, and their human orthologs are mutated in HPS3, HPS5, and HPS6 patients. These three genes encode novel proteins of unknown function. The phenotypes of Hps5/Hps5,Hps6/Hps6 and Hps3/Hps3,Hps6/Hps6 double mutant mice mimic, in coat and eye colors, in melanosome ultrastructure, and in levels of platelet dense granule serotonin, the corresponding phenotypes of single mutants. These facts suggest that the proteins encoded by these genes act within the same pathway or protein complex in vivo to regulate vesicle trafficking. Further, the Hps5 protein is destabilized within tissues of Hps3 and Hps6 mutants, as is the Hps6 protein within tissues of Hps3 and Hps5 mutants. Also, proteins encoded by these genes co-immunoprecipitate and occur in a complex of 350 kDa as determined by sucrose gradient and gel filtration analyses. Together, these results indicate that the Hps3, Hps5, and Hps6 proteins regulate vesicle trafficking to lysosome-related organelles at the physiological level as components of the BLOC-2 (biogenesis of lysosome-related organelles complex-2) protein complex and suggest that the pathogenesis and future therapies of HPS3, HPS5, and HPS6 patients are likely to be similar. Interaction of the Hps5 and Hps6 proteins within BLOC-2 is abolished by the three-amino acid deletion in the Hps6ru mutant allele, indicating that these three amino acids are important for normal BLOC-2 complex formation. Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous inherited disease affecting vesicle trafficking among lysosome-related organelles. The Hps3, Hps5, and Hps6 genes are mutated in the cocoa, ruby-eye-2, and ruby-eye mouse pigment mutants, respectively, and their human orthologs are mutated in HPS3, HPS5, and HPS6 patients. These three genes encode novel proteins of unknown function. The phenotypes of Hps5/Hps5,Hps6/Hps6 and Hps3/Hps3,Hps6/Hps6 double mutant mice mimic, in coat and eye colors, in melanosome ultrastructure, and in levels of platelet dense granule serotonin, the corresponding phenotypes of single mutants. These facts suggest that the proteins encoded by these genes act within the same pathway or protein complex in vivo to regulate vesicle trafficking. Further, the Hps5 protein is destabilized within tissues of Hps3 and Hps6 mutants, as is the Hps6 protein within tissues of Hps3 and Hps5 mutants. Also, proteins encoded by these genes co-immunoprecipitate and occur in a complex of 350 kDa as determined by sucrose gradient and gel filtration analyses. Together, these results indicate that the Hps3, Hps5, and Hps6 proteins regulate vesicle trafficking to lysosome-related organelles at the physiological level as components of the BLOC-2 (biogenesis of lysosome-related organelles complex-2) protein complex and suggest that the pathogenesis and future therapies of HPS3, HPS5, and HPS6 patients are likely to be similar. Interaction of the Hps5 and Hps6 proteins within BLOC-2 is abolished by the three-amino acid deletion in the Hps6ru mutant allele, indicating that these three amino acids are important for normal BLOC-2 complex formation. The cocoa (coa/Hps3), ruby-eye-2 (ru2/Hps5), and ruby-eye (ru/Hps6) mouse pigment genes encode novel proteins, which regulate the synthesis of lysosome-related organelles including melanosomes and platelet dense granules (1Suzuki T. Li W. Zhang Q. Novak E.K. Sviderskaya E.V. Wilson A. Bennett D.C. Roe B.A. Swank R.T. Spritz R.A. Genomics. 2001; 78: 30-37Crossref PubMed Scopus (71) Google Scholar, 2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar). Hps3, Hps5, and Hps6 mutant mice have morphologically abnormal melanosomes and decreased quantities of intragranular components of platelet dense granules (3Novak E.K. Wieland F. Jahreis G.P. Swank R.T. Biochem. Genet. 1980; 18: 549-561Crossref PubMed Scopus (14) Google Scholar, 4Novak E.K. Hui S.-W. Swank R.T. Blood. 1984; 63: 536-544Crossref PubMed Google Scholar, 5Swank R.T. Novak E.K. McGarry M.P. Rusiniak M.E. Feng L. Pigment Cell Res. 1998; 11: 60-80Crossref PubMed Scopus (176) Google Scholar, 6Swank R.T. Novak E.K. McGarry M.P. Zhang Q. Feng L. Pigment Cell Res. 2000; 13: 59-67Crossref PubMed Scopus (61) Google Scholar). Organellar trafficking abnormalities lead, in turn, to hypopigmentation of both coat and eyes and prolonged bleeding times. All three mutants are appropriate animal models for the inherited human disease Hermansky-Pudlak syndrome (HPS) 1The abbreviations used are: HPS, Hermansky-Pudlak syndrome; RPE, retinal pigment epithelia. 1The abbreviations used are: HPS, Hermansky-Pudlak syndrome; RPE, retinal pigment epithelia. (Mendelian Inheritance in Man, 203300) (7Huizing M. Gahl W.A. Curr. Mol. Med. 2002; 2: 451-467Crossref PubMed Scopus (98) Google Scholar, 8Spritz R.A. Trends Genet. 1999; 15: 337-340Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), which presents with similar abnormalities of subcellular organelles. Associated clinical symptoms of HPS include loss of visual acuity, prolonged bleeding, and lung disease due to abnormalities of melanosomes, platelet dense granules, and lysosomes, respectively. Hps3, Hps5, and Hps6 mutant mice are among at least 16 mouse models of HPS (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar, 5Swank R.T. Novak E.K. McGarry M.P. Rusiniak M.E. Feng L. Pigment Cell Res. 1998; 11: 60-80Crossref PubMed Scopus (176) Google Scholar). Human HPS patients with mutations in seven mouse HPS genes have been identified (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar, 7Huizing M. Gahl W.A. Curr. Mol. Med. 2002; 2: 451-467Crossref PubMed Scopus (98) Google Scholar, 9Li W. Zhang Q. Oiso N. Novak E.K. Gautam R. O'Brien E.P. Tinsley C.L. Blake D.J. Spritz R.A. Copeland N.G. Jenkins N.A. Amato D. Roe B.A. Starcevic M. Dell'Angelica E.C. Elliott R.W. Mishra V. Kingsmore S.F. Paylor R.E. Swank R.T. Nat. Genet. 2003; 35: 84-89Crossref PubMed Scopus (375) Google Scholar). One class of five HPS genes (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar) encodes proteins with established functions in vesicle trafficking to lysosome-related organelles in both lower and higher eukaryotes. In contrast, the second class of nine genes (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar, 10Ciciotte S.L. Gwynn B. Moriyama K. Huizing M. Gahl W.A. Bonifacino J.S. Peters L.L. Blood. 2003; 101: 4402-4407Crossref PubMed Scopus (73) Google Scholar, 11Chiang P.W. Oiso N. Gautam R. Suzuki T. Swank R.T. Spritz R.A. J. Biol. Chem. 2003; 278: 20332-20337Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which includes the Hps3, Hps5, and Hps6 genes of this report, are expressed only in higher eukaryotes and encode novel proteins with no recognizable structural motifs and whose functions are unknown. Most recently (9Li W. Zhang Q. Oiso N. Novak E.K. Gautam R. O'Brien E.P. Tinsley C.L. Blake D.J. Spritz R.A. Copeland N.G. Jenkins N.A. Amato D. Roe B.A. Starcevic M. Dell'Angelica E.C. Elliott R.W. Mishra V. Kingsmore S.F. Paylor R.E. Swank R.T. Nat. Genet. 2003; 35: 84-89Crossref PubMed Scopus (375) Google Scholar) the sandy (sdy/Hps7/Dtnbp1) gene was identified as encoding dysbindin, a dystrobrevin interacting protein (12Benson M.A. Newey S.E. Martin-Rendon E. Hawkes R. Blake D.J. J. Biol. Chem. 2001; 276: 24232-24241Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The Hps5 and Hps6 proteins directly interact in a multiprotein complex termed BLOC-2 (biogenesis of lysosome organelles complex-2) (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar). Hps3 mice have coat color (13Sweet H.O. Prochaska M. Mouse News Lett. 1985; 73: 18Google Scholar) similar to that of Hps5 and Hps6 mutants, which, in turn, are mimic mutants regarding coat and eye colors (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar, 14Silvers W.K. The Coat Colors of Mice. Springer-Verlag, New York1979: 103-104Google Scholar). These facts suggested that the function(s) of the Hps3, Hps5, and Hps6 genes are related and that they might be residents of a common protein complex. To test this hypothesis and to better understand the novel proteins of the BLOC-2 complex, we tested for epistatic interactions of the Hps3, Hps5, and Hps6 genes in doubly mutant mice and for complex formation by their protein products. Mice—Mutant mice together with normal C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were subsequently bred and maintained in the animal facilities of Roswell Park Cancer Institute. Unless indicated otherwise, the particular alleles utilized in these studies are as follows. The Hps3coa allele contains a splice site mutation resulting in a frameshift and loss of expression of the Hps3 mRNA (1Suzuki T. Li W. Zhang Q. Novak E.K. Sviderskaya E.V. Wilson A. Bennett D.C. Roe B.A. Swank R.T. Spritz R.A. Genomics. 2001; 78: 30-37Crossref PubMed Scopus (71) Google Scholar), the Hps5ru-2J allele contains a frameshift mutation that causes loss of the C-terminal third of the Hps5 protein (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar), the Hps6ru allele contains a small in-frame deletion that results in loss of three amino acids at positions 187-189 (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar). The Hps6ru-6J mutation contains a 5.3-kb intracisternal A particle element insertion that causes loss of transcript expression (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar). The Hps3coa mutation arose and is maintained on the C57BL/10J background (15Novak E.K. Sweet H.O. Prochazka M. Parentis M. Soble R. Reddington M. Cairo A. Swank R.T. Br. J. Haematol. 1988; 69: 371-378Crossref PubMed Scopus (55) Google Scholar). Both the Hps5ru-2J and Hps6ru mutations arose on the C3H inbred strain background and were subsequently transferred to and maintained as congenic mutants on the C57BL/6J inbred strain background. All mice utilized in these experiments were 2-5 months old. All procedures (mouse protocol 125M) were reviewed and approved by the Roswell Park Institutional Animal Care and Use Committee and adhered to the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Construction of Double Mutant Mice—Heterozygous F1 offspring (Hps3/+ and Hps6/+ or Hps5/+ and Hps6/+) were produced by mating of Hps3/Hps3 and Hps6/Hps6 or Hps5/Hps5 and Hps6/Hps6 mice and were, as expected, of normal black coat and eye color. F1 offspring were mated to produce an F2 generation. F2 mice doubly homozygous (Hps3/Hps3,Hps6/Hps6 or Hps5/Hps5,Hps6/Hps6) for mutant genes were verified by molecular diagnoses of genotype at each gene by PCR amplification and sequencing of normal and genomic tail DNA using appropriate primers (1Suzuki T. Li W. Zhang Q. Novak E.K. Sviderskaya E.V. Wilson A. Bennett D.C. Roe B.A. Swank R.T. Spritz R.A. Genomics. 2001; 78: 30-37Crossref PubMed Scopus (71) Google Scholar, 2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar) (sequences available upon request). Because double mutants appeared healthy and had no obvious reductions in breeding efficiency, they were mated among themselves to maintain the double mutant colonies. Because all single and double mutants are on the C57BL/6J strain background (or on the closely related C57BL/10J strain background in the case of the Hps3 mutant), contributions of background genes are essentially identical in all. Antibodies—The peptide sequence, CNQERRGKPERIHVSSE, located near the amino terminus of the Hps5 protein (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar), was conjugated to carrier protein KLH, and a polyclonal antiserum was prepared in rabbits by Covance, Denver, PA. To prepare antisera to the Hps6 protein, an expression plasmid pET 15b (Novagen) encoding the His-tagged C-terminal half (residues 1201 to 2418) of the Hps6 protein was transformed into Escherichia coli BL21 DE3 (Novagen). The Hps6 protein was expressed within inclusion bodies, solubilized with 6 m urea, and purified by Ni2+-Sepharose affinity chromatography (16Seabra M.C. Ho Y.K. Anant J.S. J. Biol. Chem. 1995; 270: 24420-24427Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) followed by dialysis successively against 4 m, 2 m, and no urea in 20 mm Tris-HCl buffer, pH 7.4. Rabbits were initially injected with 250 μg of purified His-Ruby protein followed by boosting with 125 μg bi-weekly before final collection of antisera at 100 days. Platelet Collection and Platelet Serotonin Analyses—Platelets were harvested from the peripheral blood of normal and mutant mice in the presence of sodium citrate (17Swank R.T. Reddington M. Howlett O. Novak E.K. Blood. 1991; 78: 2036-2044Crossref PubMed Google Scholar). Washed platelets were lysed in 1 ml of distilled water, counted in a Coulter Z2 particle count and size analyzer, and assayed fluorometrically for serotonin (17Swank R.T. Reddington M. Howlett O. Novak E.K. Blood. 1991; 78: 2036-2044Crossref PubMed Google Scholar). Immunoblotting—Tissue extracts were subjected to denaturing SDS gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with nonfat dry milk or ECL Advance blocking agent in phosphate-buffered saline with 0.1% Tween 20 for 1 h followed by incubation with primary antiserum at 1:1000 dilution for 1 h. After washing 1 h with blocking solution the membrane was incubated with 1:50,000 dilution of anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) for 1 h and washed with phosphate-buffered saline containing 0.1% Tween 20. Bound antibody was detected using the ECL Plus (+) system for Hps6 and ECL Advance for Hps5 (Amersham Biosciences). Blots were calibrated with Kaleidoscope prestained molecular weight standards (Bio-Rad). Monoclonal mouse anti-α tubulin (Sigma) was used as loading control. Multiple exposures confirmed that blots were exposed within a linear range. Yeast Two-hybrid Analyses—The Matchmaker GAL4 Two-Hybrid System 3 kit (Clontech) for two-hybrid analyses was used at low and high stringency as described (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar). HPS6 mutant constructs in DNA binding domain (pGBKT7) and activation domain (pGADT7) vectors were produced by deleting the three amino acids (His-Cys-Pro) at positions 187-189 from the wild type HPS6 cDNA. HPS6 alanine mutant constructs were prepared by site-directed mutagenesis by singly replacing each of these three amino acids in the wild type construct with alanine. All constructs were cloned in-frame to the DNA binding and activation domains of the Gal4 transcription factor and verified by sequencing. Plates were incubated at 30 °C for 5 days and monitored for growth and blue color by visual inspection. To verify production of construct proteins in yeast, extracts from colonies growing on low stringency plates were immunoblotted with antibodies to either Myc (goat polyclonal, 1:250 dilution; Santa Cruz Biotechnology, Inc.) or hemagglutinin (mouse monoclonal antibody, 1:1000 dilution; Berkeley Antibody Company) epitopes, which were fused in-frame to cDNAs. Electron Microscopy—Eyes were fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in spur resin as described (1Suzuki T. Li W. Zhang Q. Novak E.K. Sviderskaya E.V. Wilson A. Bennett D.C. Roe B.A. Swank R.T. Spritz R.A. Genomics. 2001; 78: 30-37Crossref PubMed Scopus (71) Google Scholar) before viewing on a Siemens 101 Electron microscope at an accelerating voltage of 80 kV. Coimmunoprecipitation—Open reading frames of Hps3, Hps5, Hps6, Hps7, Ap3b1, and pa cDNAs were fused in-frame to pCMV-Tag vectors (Myc and FLAG), and the resulting fusion constructs were verified by sequencing. Human embryonic kidney 293 cells (3 × 105) were cotransfected, using FuGENE 6 (Roche Applied Science), with epitope-tagged constructs at a ratio of 1:1, except in the cases of (a) Hps5 FLAG with Ap3b1-Myc, Hps6-Myc, or Hps3-Myc (1.8:0.2), (b) Hps7-FLAG with coa-Myc (0.5:1.5), and (c) Hps6-FLAG with Hps3-Myc (0.25:1.75). The cells were also singly transfected with Myc epitope-tagged constructs of pa, Hps3, Hps5, and Hps6. At 48 h after transfection, proteins were solubilized with 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and protease inhibitors for 1 h at 4 °C. Samples were immunoprecipitated by incubating them for 1 h at 4 °C with FLAG M2 antibody-conjugated agarose (Sigma) and washed with agarose beads three times with 0.5 m Tris-HCl, pH 7.4, plus 1.5 m NaCl. Bound proteins were eluted by treating the samples for 5 min at 95 °C with denaturing Laemmli buffer, and blots of 8% SDS-PAGE gels were analyzed with either rabbit polyclonal antibody against FLAG (1:1,000 dilution; Affinity Bio-Reagents) or with goat polyclonal antibody against Myc (1:250 dilution; Santa Cruz Biotechnology, Inc.). Horseradish peroxidase-linked donkey antibody against rabbit immunoglobulin G (1:5000 dilution; Amersham Biosciences) was used as a secondary antibody for FLAG blots, and horseradish peroxidase-linked bovine antibody against goat immunoglobulin G (1:5000 dilution; Santa Cruz Biotechnology, Inc.) was used as a secondary antibody for Myc blots. Blots were treated with the enhanced chemiluminescence reagent (ECL Plus (+); Amersham Biosciences) and exposed for 1 min. Size-exclusion Chromatography and Sedimentation Velocity Analysis—Cytosolic extracts from the liver of normal and mutant mice were prepared by homogenization in detergent-free Tris buffer (0.3 M Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm MgCl2, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 mg/liter leupeptin, 5 mg/liter aprotinin and 1 mg/liter pepstatin A), using a Dounce homogenizer, followed by centrifugation at 5000 × g for 5 min and then at 120,000 × g for 90 min, at 4 °C. Size-exclusion chromatography was performed as described (18Falcon-Perez J.M. Starcevic M. Gautam R. Dell'Angelica E.C. J. Biol. Chem. 2002; 277: 28191-28199Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Sedimentation velocity analysis was carried out by ultracentrifugation of cytosolic extract (0.2 ml, ∼5 mg total protein) loaded on top of a linear 5-20% (w/v) sucrose gradient prepared in detergent-free Tris buffer (12 ml), for 13 h at 39,000 rpm on a SW41 rotor (Beckman Coulter). Fractions were collected from the bottom of the tube. Fractions resulting from both the size-exclusion chromatography and sedimentation velocity experiments were analyzed by immunoblotting using antibodies to Hps5 and Hps6 proteins. Cytosolic liver extracts from Hps5ru-2J and Hps6ru-6J null mutant mice were analyzed in parallel to confirm the identity of the Hps5 and Hps6 protein bands, respectively. In Vivo Physiological Interactions of Hps3, Hps5, and Hps6 Proteins in the Production of Lysosome-related Organelles in Doubly Mutant Mice—To test for interactions between (a) the Hps5 and Hps6 gene products and (b) the Hps3 and Hps6 gene products at the physiological level, appropriate double mutant mice (i.e. homozygous for mutant genes at two of these HPS loci) were bred, verified (see “Experimental Procedures”), and analyzed for abnormalities of melanosomes and platelet dense granules, the lysosome-related organelles most severely affected in Hps5 and Hps6 mutants. The coat and eye colors of Hps5/Hps5,Hps6/Hps6 double mutants are hypopigmented in comparison to C57BL/6J controls and identical (Fig. 1A) to those of the Hps5/Hps5 and Hps6/Hps6 single mutants (which themselves exhibit mimic phenotypes) (see Fig. 1A) suggesting a common abnormality in melanosomes, the subcellular organelle that imparts coat and eye coloration. In both single and double mutants, coat colors are the classical (14Silvers W.K. The Coat Colors of Mice. Springer-Verlag, New York1979: 103-104Google Scholar) ruby color. Eye color in all is a light pink in offspring less than 1 week of age (not shown). This deepens in adults to a dark ruby eye color, distinguishable from the black eyes of normal C57BL/6J mice only when closely observed with intense light. The detailed mimicry in pigmentation and melanosomal properties of the Hps5 and Hps6 single and double mutants extends to the ultrastructural level in melanosomes of the retinal pigment epithelia (RPE) and choroids of the eye (Fig. 2). Quantitatively, there are major and similar reductions in numbers of melanosomes of the RPE in single and double mutants. Likewise, all mutant abnormalities are qualitatively concordant. The few remaining melanosomes within the RPE are often of unusual morphology, and some appear to be end-stage degradative organelles. All mutants contain larger numbers of choroidal than RPE melanosomes. However, all choroidal melanosomes are smaller than those of the control C57BL/6J and often have uneven “ragged” edges. The most conspicuous feature of the choroids of all single and double mutants is clumping (Fig. 2) of melanosomes within a single membrane-limited body, a morphological feature not observed in other mouse HPS mutants. Identical conclusions apply to Hps3 and Hps6 single and double mutant mice, which were likewise bred and analyzed for abnormalities of coat and eye color and melanosome ultrastructure. Coat and eye colors of single (Hps3/Hps3 and Hps6/Hps6) mimic those of double (Hps3/Hps3,Hps6/Hps6) mutants (Fig. 1B). Likewise, the degree of hypopigmentation and the color of the eyes of newborn (not shown) Hps3/Hps3 and Hps3/Hps3,Hps6/Hps6 mutants mimic those of the above-described newborn Hps5/Hps5 and Hps6/Hps6 mice. At the ultrastructural level melanosomes of the RPE and choroid of double mutants are indistinguishable from those of all three single mutants and include the distinctive melanosome aggregates (Fig. 2) within the choroids. Taken together, these analyses of coat color and melanosome ultrastructure in three single and two double mutants suggest a common defect in the synthesis and/or processing of melanosomes in Hps3, Hps5, and Hps6 mutants. Gene dosage does not affect the mutant phenotype, because by visual examination, the Hps5/+,Hps6/Hps6; Hps5/Hps5, Hps6/+; Hps3/+,Hps6/Hps6, and Hps3/Hps3,Hps6/+ mice (not shown) are identical in coat and eye color to the original single mutant mice and to each other. Both single and double mutant mice appear healthy and robust for at least eight months of age. A lysosome-related organelle invariably affected in all HPS patients and animal models is the platelet dense granule (5Swank R.T. Novak E.K. McGarry M.P. Rusiniak M.E. Feng L. Pigment Cell Res. 1998; 11: 60-80Crossref PubMed Scopus (176) Google Scholar, 7Huizing M. Gahl W.A. Curr. Mol. Med. 2002; 2: 451-467Crossref PubMed Scopus (98) Google Scholar, 8Spritz R.A. Trends Genet. 1999; 15: 337-340Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Typically, platelet dense granules are either missing or greatly reduced in number. Alternatively, granules may be present, but are “empty,” a condition documented in Hps3, Hps5, and Hps6 mutants (15Novak E.K. Sweet H.O. Prochazka M. Parentis M. Soble R. Reddington M. Cairo A. Swank R.T. Br. J. Haematol. 1988; 69: 371-378Crossref PubMed Scopus (55) Google Scholar, 19Reddington M. Novak E.K. Hurley E. Medda C. McGarry M.P. Swank R.T. Blood. 1987; 69: 1300-1306Crossref PubMed Google Scholar). Either condition leads to functionally abnormal platelets and prolonged bleeding times. The serotonin concentrations within platelet dense granules were indistinguishable among all single and double mutants, being greatly depressed to 6-8% that of normal C57BL/6J controls. Consistent with these findings, all single and double mutants had bleeding times (not shown) greater than 15 min compared with the 2-4-min times of C57BL/6J controls. Combined, these several mimic effects on melanosomes and platelet dense granules suggest that the Hps3, Hps5, and Hps6 genes regulate the synthesis of lysosome-related organelles by a common mechanism at the physiological level. Test for Destabilization of HPS5 and HPS6 Proteins in Extracts of Other Hps Mutants—The above mimic effects of the Hps3, Hps5, and Hps6 genes suggested possible co-residence of their protein products within a common protein complex. An indication of residence of two proteins within a common protein complex is destabilization of the partner protein within cells derived from mutants lacking one of the proteins, as loss of one member of a protein complex often leads to destabilization of other members of that complex (18Falcon-Perez J.M. Starcevic M. Gautam R. Dell'Angelica E.C. J. Biol. Chem. 2002; 277: 28191-28199Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 20Moriyama K. Bonifacino J.S. Traffic. 2002; 3: 666-677Crossref PubMed Scopus (62) Google Scholar). Accordingly, polyclonal antibodies to the Hps5 and Hps6 proteins were produced, and levels of these proteins were analyzed by Western blotting in tissues of 14 mouse HPS mutants and alleles to determine whether mutations in other HPS genes affected their concentrations (Fig. 3). Consistent with its residence, together with the Hps6 protein, within the BLOC-2 complex (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar), the Hps5 protein exhibits destabilization within spleen and lung extracts of the Hps6ru-6J null mutant (Fig. 3). Significant destabilization of Hps5 protein is also apparent in extracts of the Hps3 null mutant. Its concentration is also, as expected, depressed in extracts of single and double mutants containing the Hps5ru2-J allele, which have undetectable Hps5 protein levels, an expected result given the frameshift null mutation within this allele (2Zhang Q. Zhao B. Li W. Oiso N. Novak E.K. Rusiniak M.E. Gautam R. Chintala S. O'Brien E.P. Zhang Y. Roe B.A. Elliott R.W. Eicher E.M. Liang P. Kratz C. Legius E. Spritz R.A. O'Sullivan T.N. Copeland N.G. Jenkins N.A. Swank R.T. Nat. Genet. 2003; 33: 145-153Crossref PubMed Scopus (163) Google Scholar). Similar results were observed in heart extracts (not shown). No significant destabilization was apparent in any of the remaining 11 HPS mutants or the misty hypopigmentation mutant. Similarly, in regard to possible residence within a common protein complex, there was a notable
Hermansky–Pudlak syndrome
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Organelle biogenesis
Melanosome
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Hermansky–Pudlak syndrome
Organelle
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Lysosomes are organelles that receive external cargo through phagocytosis and endocytosis, and internal cargo through autophagy, followed by degradation in the acidic and hydrolase rich lumen and redistribution of substrates for maintaining cellular integrity. Lysosomes undergo homotypic or heterotypic repeated fusion and fission or kiss and run cycles with other organelles to exchange and receive cargo, as well as maintain lysosome number and size. Lysosome membranes display the phosphoinositide lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) synthesized by the lipid kinase PIKfyve. PtdIns(3,5)P₂ act as a signalling lipid on lysosomes to regulate maturation of endosomes, phagosomes and autophagosomes maturation by fusing with lysosomes, and recycling from the lysosomal lumen, lysosome ion channel activity, and lysosome-associated actin turnover. Of these defects, the most dramatic phenotype of PtdIns(3,5)P₂ depletion from PIKfyve inhibition is the appearance of enlarged lysosomes. Our work demonstrates that PtdIns(3,5)P₂ is an important regulator of lysosome size and number by governing the balance between lysosome fusion and fission and/or kiss and run. Depletion of PtdIns(3,5)P₂ arrests lysosome fission disrupting the balance between the continuous fusion and fission cycle, leading to lysosome coalescence and causing lysosome enlargement and reduction in their numbers. Microtubules, cytoskeletal tracks for lysosome positioning, and associated motor protein complexes, kinesin-1 and dynein, regulate lysosome coalescence during PIKfyve inhibition. Our experimental observations revealed ROS as a novel regulator of lysosome fusion and fission. Specifically, ROS arrested lysosome enlargement from acute PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS produced and/or site of ROS synthesis, lysosome dynamics are affected distinctly. H₂O₂ impaired lysosome mobility to arrest coalescence. However, superoxide generated from mitochondrial ETC complex 1, or thioredoxin reductase, or glutathione inhibition through rotenone, or CDNB, or MCB respectively depolymerised microtubules without affecting mobility. Instead, superoxide generation through pharmacological manipulations promoted actin clearance from lysosomes, which otherwise accumulate on lysosomes to hinder fission upon PIKfyve inhibition, to promote fission. Indeed, actin depolymerisation arrested lysosome enlargement during acute PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation, further indicative of ROS stimulating lysosome fission through actin clearance.
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Introduction: Autophagy is a self-destructive, lysosomal catabolic pathway and is known from several decades as an adaptation to starvation. Basal autophagy is extremely important in turn-over of long lived proteins. Defective or severely altered macroautophagy has been reported and has been implicated in the pathogenesis of many lysosome related disorders. Hermansky-Pudlak syndrome (HPS) is one such lysosome realated disorder, affecting several lysosome-related organelles of the body. Several HPS mutations have been identified so far, but patients only with HPS types -1 & -4 develop pulmonary fibrosis called Hermansky-Pudlak syndrome associated Interstitial Pneumonia (HPSIP), the main reason of death in such patients. HPSIP lungs typically show enlarged alveolar typeII cells (AECII) with giant lamellar bodies, the lysosome related organelles of AECII. We recently reported spontaneous development of lung fibrosis in a mouse model of HPSIP (HPS1/2 mice), severe surfactant accumulation and apoptosis of AECII due to severe lysosomal stress and ER stress in these mice as well as in human HPS1.
Hermansky–Pudlak syndrome
Lipofuscin
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Abstract Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host’ multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca 2+ -dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca 2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1’s ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.
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Abstract The bidirectional movement of lysosomes on microtubule tracks regulates their whole-cell spatial arrangement. Arl8b, a small GTP-binding (G) protein, promotes lysosome anterograde trafficking mediated by kinesin-1. Herein, we report an Arl8b effector, RUFY3, which regulates the retrograde transport of lysosomes. We show that RUFY3 interacts with the JIP4-dynein-dynactin complex and facilitates Arl8b association with the retrograde motor complex. Accordingly, RUFY3 knockdown disrupts the positioning of Arl8b-positive endosomes and reduces Arl8b colocalization with Rab7-marked late endosomal compartments. Moreover, we find that RUFY3 regulates nutrient-dependent lysosome distribution, although autophagosome-lysosome fusion and autophagic cargo degradation are not impaired upon RUFY3 depletion. Interestingly, lysosome size is significantly reduced in RUFY3 depleted cells, which could be rescued by inhibition of the lysosome reformation regulatory factor PIKFYVE. These findings suggest a model in which the perinuclear cloud arrangement of lysosomes regulates both the positioning and size of these proteolytic compartments.
Colocalization
Small GTPase
Autophagosome
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Both macroautophagy/autophagy and extracellular vesicle (EV) secretion pathways converge upon the endolysosome system. Although lysosome impairment leads to defects in autophagic degradation, the impact of such dysfunction on EV secretion remains poorly understood. Recently, we uncovered a novel secretory autophagy pathway that employs EVs and nanoparticles (EVPs) for the secretion of autophagy cargo receptors outside the cell when either autophagosome maturation or lysosomal function is blocked. We term this process secretory autophagy during lysosome inhibition (SALI). SALI functionally requires multiple steps in classical autophagosome formation and the small GTPase RAB27A. Because the intracellular accumulation of autophagy cargo receptors perturbs cell signaling and quality control pathways, we propose that SALI functions as a failsafe mechanism to preserve protein and cellular homeostasis when autophagic or lysosomal degradation is impaired.
Autophagosome
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Lysosomes are organelles that receive external cargo through phagocytosis and endocytosis, and internal cargo through autophagy, followed by degradation in the acidic and hydrolase rich lumen and redistribution of substrates for maintaining cellular integrity. Lysosomes undergo homotypic or heterotypic repeated fusion and fission or kiss and run cycles with other organelles to exchange and receive cargo, as well as maintain lysosome number and size. Lysosome membranes display the phosphoinositide lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) synthesized by the lipid kinase PIKfyve. PtdIns(3,5)P₂ act as a signalling lipid on lysosomes to regulate maturation of endosomes, phagosomes and autophagosomes maturation by fusing with lysosomes, and recycling from the lysosomal lumen, lysosome ion channel activity, and lysosome-associated actin turnover. Of these defects, the most dramatic phenotype of PtdIns(3,5)P₂ depletion from PIKfyve inhibition is the appearance of enlarged lysosomes. Our work demonstrates that PtdIns(3,5)P₂ is an important regulator of lysosome size and number by governing the balance between lysosome fusion and fission and/or kiss and run. Depletion of PtdIns(3,5)P₂ arrests lysosome fission disrupting the balance between the continuous fusion and fission cycle, leading to lysosome coalescence and causing lysosome enlargement and reduction in their numbers. Microtubules, cytoskeletal tracks for lysosome positioning, and associated motor protein complexes, kinesin-1 and dynein, regulate lysosome coalescence during PIKfyve inhibition. Our experimental observations revealed ROS as a novel regulator of lysosome fusion and fission. Specifically, ROS arrested lysosome enlargement from acute PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS produced and/or site of ROS synthesis, lysosome dynamics are affected distinctly. H₂O₂ impaired lysosome mobility to arrest coalescence. However, superoxide generated from mitochondrial ETC complex 1, or thioredoxin reductase, or glutathione inhibition through rotenone, or CDNB, or MCB respectively depolymerised microtubules without affecting mobility. Instead, superoxide generation through pharmacological manipulations promoted actin clearance from lysosomes, which otherwise accumulate on lysosomes to hinder fission upon PIKfyve inhibition, to promote fission. Indeed, actin depolymerisation arrested lysosome enlargement during acute PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation, further indicative of ROS stimulating lysosome fission through actin clearance.
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