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Human Vam6p promotes lysosome clustering and fusion in vivo.

Caplan S, Hartnell LM, Aguilar RC, Naslavsky N, Bonifacino JS - J. Cell Biol. (2001)

Bottom Line: This effect is reminiscent of that caused by expression of a constitutively activated Rab7.However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7.Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch at the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH(2) terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2-3 microm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

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Sequence homology, domain organization, and expression of hVam6p. (A) Full-length amino acid sequences of human (Homo sapiens; Hs) Vam6p (hVam6p), a D. melanogaster (Dm) homologue, and a C. elegans (Ce) homologue were aligned together with residues 451–1049 of S. cerevisiae (Sc) Vam6p/Vps39p, using the ClustalW Multiple Sequences Alignment software (available at the European Bioinformatics Institute website http://www2.ebi.ac.uk/clustalw/) and shaded using the BOXSHADE program. Identical and similar residues are indicated by black and gray shading, respectively. The blue line denotes the hypothetical hVam6p CNH domain, which is conserved in Dm and Ce but not Sc. The brown line indicates the position of the hypothetical hVam6p CLH domain, which is conserved in all four orthologues. Domains were identified by Pfam HMM database searches (available from Washington University at http://pfam.wustl.edu/hmmsearch.shtml) (B) Domain organization of Vam6p/Vps39p family members. Specific domains are color-coded and regions of significant homology to hVam6p are shown in blue (CNH), brown (CLH), and green. GenBank/EMBL/DDBJ accession numbers are as follows: Hs Vam6p/Vps39p (AF280814), Ds Vam6p (AAF55525), Ce Vam6p (T24712), Sp Vam6p (T38314), Hs TRAP-1 (XP 002298), Sc Vam6p/Vps39p (BAA11758), Sc Rom1p (S64365), Hs Traf2 and NCK-interacting kinase (TNIK) (AF172270), Hs NCK-interacting kinase–like (AAC83079), Hs Vam2p/Vps41p (P49754), Sc Vam2p/Vps41p (BAA19071). Note that whereas the S. cerevisiae CLH domain has previously been situated between residues 716 and 900 (Wurmser et al., 2000); our domain analysis, using the Pfam program, positions it between residues 512 and 676. DEP is a domain found in Dishevelled, Egl-10, and Pleckstrin. (C) Analysis of hVam6 mRNA expression in different human tissues. Northern blots with mRNA from various human tissues were analyzed with a 32P-labeled probe specific for the complete hVam6 mRNA. The positions of RNA size markers are indicated.
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fig1: Sequence homology, domain organization, and expression of hVam6p. (A) Full-length amino acid sequences of human (Homo sapiens; Hs) Vam6p (hVam6p), a D. melanogaster (Dm) homologue, and a C. elegans (Ce) homologue were aligned together with residues 451–1049 of S. cerevisiae (Sc) Vam6p/Vps39p, using the ClustalW Multiple Sequences Alignment software (available at the European Bioinformatics Institute website http://www2.ebi.ac.uk/clustalw/) and shaded using the BOXSHADE program. Identical and similar residues are indicated by black and gray shading, respectively. The blue line denotes the hypothetical hVam6p CNH domain, which is conserved in Dm and Ce but not Sc. The brown line indicates the position of the hypothetical hVam6p CLH domain, which is conserved in all four orthologues. Domains were identified by Pfam HMM database searches (available from Washington University at http://pfam.wustl.edu/hmmsearch.shtml) (B) Domain organization of Vam6p/Vps39p family members. Specific domains are color-coded and regions of significant homology to hVam6p are shown in blue (CNH), brown (CLH), and green. GenBank/EMBL/DDBJ accession numbers are as follows: Hs Vam6p/Vps39p (AF280814), Ds Vam6p (AAF55525), Ce Vam6p (T24712), Sp Vam6p (T38314), Hs TRAP-1 (XP 002298), Sc Vam6p/Vps39p (BAA11758), Sc Rom1p (S64365), Hs Traf2 and NCK-interacting kinase (TNIK) (AF172270), Hs NCK-interacting kinase–like (AAC83079), Hs Vam2p/Vps41p (P49754), Sc Vam2p/Vps41p (BAA19071). Note that whereas the S. cerevisiae CLH domain has previously been situated between residues 716 and 900 (Wurmser et al., 2000); our domain analysis, using the Pfam program, positions it between residues 512 and 676. DEP is a domain found in Dishevelled, Egl-10, and Pleckstrin. (C) Analysis of hVam6 mRNA expression in different human tissues. Northern blots with mRNA from various human tissues were analyzed with a 32P-labeled probe specific for the complete hVam6 mRNA. The positions of RNA size markers are indicated.

Mentions: A novel human cDNA encoding a protein homologous to the S. cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p (Nakamura et al., 1997) was identified through searches of DNA databases and 5′ RACE-PCR (Fig. 1 A). This cDNA encodes a protein designated hVam6p of 886 amino acid residues and a predicted molecular mass of ∼100 kD. hVam6p bears 26% identity and 42% overall amino acid sequence homology to the COOH-terminal region of S. cerevisiae Vam6p (Fig. 1, A and B). Homologues of hVam6p were also identified in Drosophila melanogaster (35% identity, 53% homology), Caenorhabditis elegans (30% identity, 50% homology) (Fig. 1, A and B), and Schizosaccharomyces pombe (24% identity, 41% homology) (Fig. 1 B). Our searches also identified a second human homologue of S. cerevisiae Vam6p that had already been characterized as the TGF-β receptor-I–associated protein-1 (TRAP-1) (21% identity, 40% homology) (Charng et al., 1998) (Fig. 1 B).


Human Vam6p promotes lysosome clustering and fusion in vivo.

Caplan S, Hartnell LM, Aguilar RC, Naslavsky N, Bonifacino JS - J. Cell Biol. (2001)

Sequence homology, domain organization, and expression of hVam6p. (A) Full-length amino acid sequences of human (Homo sapiens; Hs) Vam6p (hVam6p), a D. melanogaster (Dm) homologue, and a C. elegans (Ce) homologue were aligned together with residues 451–1049 of S. cerevisiae (Sc) Vam6p/Vps39p, using the ClustalW Multiple Sequences Alignment software (available at the European Bioinformatics Institute website http://www2.ebi.ac.uk/clustalw/) and shaded using the BOXSHADE program. Identical and similar residues are indicated by black and gray shading, respectively. The blue line denotes the hypothetical hVam6p CNH domain, which is conserved in Dm and Ce but not Sc. The brown line indicates the position of the hypothetical hVam6p CLH domain, which is conserved in all four orthologues. Domains were identified by Pfam HMM database searches (available from Washington University at http://pfam.wustl.edu/hmmsearch.shtml) (B) Domain organization of Vam6p/Vps39p family members. Specific domains are color-coded and regions of significant homology to hVam6p are shown in blue (CNH), brown (CLH), and green. GenBank/EMBL/DDBJ accession numbers are as follows: Hs Vam6p/Vps39p (AF280814), Ds Vam6p (AAF55525), Ce Vam6p (T24712), Sp Vam6p (T38314), Hs TRAP-1 (XP 002298), Sc Vam6p/Vps39p (BAA11758), Sc Rom1p (S64365), Hs Traf2 and NCK-interacting kinase (TNIK) (AF172270), Hs NCK-interacting kinase–like (AAC83079), Hs Vam2p/Vps41p (P49754), Sc Vam2p/Vps41p (BAA19071). Note that whereas the S. cerevisiae CLH domain has previously been situated between residues 716 and 900 (Wurmser et al., 2000); our domain analysis, using the Pfam program, positions it between residues 512 and 676. DEP is a domain found in Dishevelled, Egl-10, and Pleckstrin. (C) Analysis of hVam6 mRNA expression in different human tissues. Northern blots with mRNA from various human tissues were analyzed with a 32P-labeled probe specific for the complete hVam6 mRNA. The positions of RNA size markers are indicated.
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Related In: Results  -  Collection

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fig1: Sequence homology, domain organization, and expression of hVam6p. (A) Full-length amino acid sequences of human (Homo sapiens; Hs) Vam6p (hVam6p), a D. melanogaster (Dm) homologue, and a C. elegans (Ce) homologue were aligned together with residues 451–1049 of S. cerevisiae (Sc) Vam6p/Vps39p, using the ClustalW Multiple Sequences Alignment software (available at the European Bioinformatics Institute website http://www2.ebi.ac.uk/clustalw/) and shaded using the BOXSHADE program. Identical and similar residues are indicated by black and gray shading, respectively. The blue line denotes the hypothetical hVam6p CNH domain, which is conserved in Dm and Ce but not Sc. The brown line indicates the position of the hypothetical hVam6p CLH domain, which is conserved in all four orthologues. Domains were identified by Pfam HMM database searches (available from Washington University at http://pfam.wustl.edu/hmmsearch.shtml) (B) Domain organization of Vam6p/Vps39p family members. Specific domains are color-coded and regions of significant homology to hVam6p are shown in blue (CNH), brown (CLH), and green. GenBank/EMBL/DDBJ accession numbers are as follows: Hs Vam6p/Vps39p (AF280814), Ds Vam6p (AAF55525), Ce Vam6p (T24712), Sp Vam6p (T38314), Hs TRAP-1 (XP 002298), Sc Vam6p/Vps39p (BAA11758), Sc Rom1p (S64365), Hs Traf2 and NCK-interacting kinase (TNIK) (AF172270), Hs NCK-interacting kinase–like (AAC83079), Hs Vam2p/Vps41p (P49754), Sc Vam2p/Vps41p (BAA19071). Note that whereas the S. cerevisiae CLH domain has previously been situated between residues 716 and 900 (Wurmser et al., 2000); our domain analysis, using the Pfam program, positions it between residues 512 and 676. DEP is a domain found in Dishevelled, Egl-10, and Pleckstrin. (C) Analysis of hVam6 mRNA expression in different human tissues. Northern blots with mRNA from various human tissues were analyzed with a 32P-labeled probe specific for the complete hVam6 mRNA. The positions of RNA size markers are indicated.
Mentions: A novel human cDNA encoding a protein homologous to the S. cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p (Nakamura et al., 1997) was identified through searches of DNA databases and 5′ RACE-PCR (Fig. 1 A). This cDNA encodes a protein designated hVam6p of 886 amino acid residues and a predicted molecular mass of ∼100 kD. hVam6p bears 26% identity and 42% overall amino acid sequence homology to the COOH-terminal region of S. cerevisiae Vam6p (Fig. 1, A and B). Homologues of hVam6p were also identified in Drosophila melanogaster (35% identity, 53% homology), Caenorhabditis elegans (30% identity, 50% homology) (Fig. 1, A and B), and Schizosaccharomyces pombe (24% identity, 41% homology) (Fig. 1 B). Our searches also identified a second human homologue of S. cerevisiae Vam6p that had already been characterized as the TGF-β receptor-I–associated protein-1 (TRAP-1) (21% identity, 40% homology) (Charng et al., 1998) (Fig. 1 B).

Bottom Line: This effect is reminiscent of that caused by expression of a constitutively activated Rab7.However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7.Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch at the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH(2) terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2-3 microm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Show MeSH