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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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Homooligomerization of hVam6p. (A) The S. cerevisiae yeast strain AH109 was cotransformed with the following GAL4ad fusion constructs: GAL4ad–hVam6p, GAL4ad–Rab7 Q67L, and GAL4ad–pVA3 (murine p53 control), together with the GAL4bd fusion constructs GAL4bd–hVam6p, GAL4bd–RILP, and GAL4bd–pTD1 (SV40 large T-antigen control). Cotransformants were assayed for growth on nonselective (+His) and selective (−His) media. (B) Sedimentation velocity analysis of hVam6p from [35S]methionine–labeled HeLa cells. The cell extract was run on a 4–20% sucrose gradient, and fractions were analyzed by sequential immunoprecipitations. The fractions were first cleared with an irrelevant antibody and then subjected to immunoprecipitation with a second irrelevant antibody (mouse monoclonal anti-Myc). The fractions were then immunoprecipitated with a mouse monoclonal antibody to the HA epitope. Fractions from the anti-Myc (control, top) and anti-HA (bottom) immunoprecipitations were then resolved by 4–20% gradient SDS-PAGE. Thin arrows denote markers visualized by Coomassie blue staining of separated fractions subjected to the same gradient conditions (albumin and catalase). The thick arrow denotes the position of AP-2, as visualized by immunoblotting of 10% of the labeled fractions with anti–AP-2 antibody (100/2) upon completion of the immunoprecipitations.
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fig8: Homooligomerization of hVam6p. (A) The S. cerevisiae yeast strain AH109 was cotransformed with the following GAL4ad fusion constructs: GAL4ad–hVam6p, GAL4ad–Rab7 Q67L, and GAL4ad–pVA3 (murine p53 control), together with the GAL4bd fusion constructs GAL4bd–hVam6p, GAL4bd–RILP, and GAL4bd–pTD1 (SV40 large T-antigen control). Cotransformants were assayed for growth on nonselective (+His) and selective (−His) media. (B) Sedimentation velocity analysis of hVam6p from [35S]methionine–labeled HeLa cells. The cell extract was run on a 4–20% sucrose gradient, and fractions were analyzed by sequential immunoprecipitations. The fractions were first cleared with an irrelevant antibody and then subjected to immunoprecipitation with a second irrelevant antibody (mouse monoclonal anti-Myc). The fractions were then immunoprecipitated with a mouse monoclonal antibody to the HA epitope. Fractions from the anti-Myc (control, top) and anti-HA (bottom) immunoprecipitations were then resolved by 4–20% gradient SDS-PAGE. Thin arrows denote markers visualized by Coomassie blue staining of separated fractions subjected to the same gradient conditions (albumin and catalase). The thick arrow denotes the position of AP-2, as visualized by immunoblotting of 10% of the labeled fractions with anti–AP-2 antibody (100/2) upon completion of the immunoprecipitations.

Mentions: A novel Rab7 effector termed RILP (Cantalupo et al., 2001) has recently been identified through its binding to the constitutively active Rab7 Q67L and has been demonstrated to mediate the effects of Rab7 on lysosome biogenesis. To determine whether hVam6p interacts with either Rab7 Q67L or RILP, we used a yeast two-hybrid approach. As expected, Rab7 Q67L and RILP interacted with one another (Cantalupo et al., 2001; and Fig. 8 A). However, neither Rab7 Q67L nor RILP displayed an interaction with hVam6p, consistent with the idea that hVam6p acts independently of Rab7. A strong interaction was observed upon coexpression of GAL4 transcription activation domain (GAL4ad)–hVam6p and GAL4 DNA–binding domain (GAL4bd)–hVam6p (Fig. 8 A), however, suggesting that hVam6p may be able to self-assemble.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

Homooligomerization of hVam6p. (A) The S. cerevisiae yeast strain AH109 was cotransformed with the following GAL4ad fusion constructs: GAL4ad–hVam6p, GAL4ad–Rab7 Q67L, and GAL4ad–pVA3 (murine p53 control), together with the GAL4bd fusion constructs GAL4bd–hVam6p, GAL4bd–RILP, and GAL4bd–pTD1 (SV40 large T-antigen control). Cotransformants were assayed for growth on nonselective (+His) and selective (−His) media. (B) Sedimentation velocity analysis of hVam6p from [35S]methionine–labeled HeLa cells. The cell extract was run on a 4–20% sucrose gradient, and fractions were analyzed by sequential immunoprecipitations. The fractions were first cleared with an irrelevant antibody and then subjected to immunoprecipitation with a second irrelevant antibody (mouse monoclonal anti-Myc). The fractions were then immunoprecipitated with a mouse monoclonal antibody to the HA epitope. Fractions from the anti-Myc (control, top) and anti-HA (bottom) immunoprecipitations were then resolved by 4–20% gradient SDS-PAGE. Thin arrows denote markers visualized by Coomassie blue staining of separated fractions subjected to the same gradient conditions (albumin and catalase). The thick arrow denotes the position of AP-2, as visualized by immunoblotting of 10% of the labeled fractions with anti–AP-2 antibody (100/2) upon completion of the immunoprecipitations.
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Related In: Results  -  Collection

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fig8: Homooligomerization of hVam6p. (A) The S. cerevisiae yeast strain AH109 was cotransformed with the following GAL4ad fusion constructs: GAL4ad–hVam6p, GAL4ad–Rab7 Q67L, and GAL4ad–pVA3 (murine p53 control), together with the GAL4bd fusion constructs GAL4bd–hVam6p, GAL4bd–RILP, and GAL4bd–pTD1 (SV40 large T-antigen control). Cotransformants were assayed for growth on nonselective (+His) and selective (−His) media. (B) Sedimentation velocity analysis of hVam6p from [35S]methionine–labeled HeLa cells. The cell extract was run on a 4–20% sucrose gradient, and fractions were analyzed by sequential immunoprecipitations. The fractions were first cleared with an irrelevant antibody and then subjected to immunoprecipitation with a second irrelevant antibody (mouse monoclonal anti-Myc). The fractions were then immunoprecipitated with a mouse monoclonal antibody to the HA epitope. Fractions from the anti-Myc (control, top) and anti-HA (bottom) immunoprecipitations were then resolved by 4–20% gradient SDS-PAGE. Thin arrows denote markers visualized by Coomassie blue staining of separated fractions subjected to the same gradient conditions (albumin and catalase). The thick arrow denotes the position of AP-2, as visualized by immunoblotting of 10% of the labeled fractions with anti–AP-2 antibody (100/2) upon completion of the immunoprecipitations.
Mentions: A novel Rab7 effector termed RILP (Cantalupo et al., 2001) has recently been identified through its binding to the constitutively active Rab7 Q67L and has been demonstrated to mediate the effects of Rab7 on lysosome biogenesis. To determine whether hVam6p interacts with either Rab7 Q67L or RILP, we used a yeast two-hybrid approach. As expected, Rab7 Q67L and RILP interacted with one another (Cantalupo et al., 2001; and Fig. 8 A). However, neither Rab7 Q67L nor RILP displayed an interaction with hVam6p, consistent with the idea that hVam6p acts independently of Rab7. A strong interaction was observed upon coexpression of GAL4 transcription activation domain (GAL4ad)–hVam6p and GAL4 DNA–binding domain (GAL4bd)–hVam6p (Fig. 8 A), however, suggesting that hVam6p may be able to self-assemble.

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
Related in: MedlinePlus