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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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Association of hVam6p with clusters of lysosomes and late endosomes. HeLa cells were transiently transfected with HA–hVam6p and processed for immunoelectron microscopy. Ultra-thin frozen sections were labeled with antibodies to HA (to detect hVam6p) and either endogenous lamp-2 (A) or endogenous CI-MPR (B). Bound antibodies were detected using conjugated protein A–gold. Arrows indicate the localization of hVam6p to a halo around the membranes of lysosomes and late endosomes (A and B, 10-nm gold particles), whereas arrowheads indicate the localization of lamp-2 (A, 15-nm gold particles) or CI-MPR (B, 15-nm gold particles), and the asterisk marks a structure labeled only for hVam6p. G, Golgi. Bars, 0.2 μm.
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fig3: Association of hVam6p with clusters of lysosomes and late endosomes. HeLa cells were transiently transfected with HA–hVam6p and processed for immunoelectron microscopy. Ultra-thin frozen sections were labeled with antibodies to HA (to detect hVam6p) and either endogenous lamp-2 (A) or endogenous CI-MPR (B). Bound antibodies were detected using conjugated protein A–gold. Arrows indicate the localization of hVam6p to a halo around the membranes of lysosomes and late endosomes (A and B, 10-nm gold particles), whereas arrowheads indicate the localization of lamp-2 (A, 15-nm gold particles) or CI-MPR (B, 15-nm gold particles), and the asterisk marks a structure labeled only for hVam6p. G, Golgi. Bars, 0.2 μm.

Mentions: The localization of hVam6p in transfected HeLa cells was further examined by immunoelectron microscopy of ultrathin frozen sections (Fig. 3) . hVam6p was found to be associated with an electron dense halo surrounding 0.2–0.6 μm vesicles that were part of large clusters (Fig. 3 A, 10-nm gold particles, arrows). All of these vesicles were also labeled for the lysosomal membrane protein, lamp-2 (Fig. 3 A, 15-nm gold particles, arrowheads). Although many hVam6p-coated vesicles did not contain cation-independent mannose 6-phosphate receptor (CI-MPR) (Fig 3 B, *), others contained both hVam6p (Fig. 3 B, 10-nm gold particles, arrows) and CI-MPR (Fig. 3 B, 15-nm gold particles, arrowhead), and some had the appearance of multivesicular bodies. Together, these observations suggest that hVam6p associates with and induces clustering of lysosomes and late endosomes.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

Association of hVam6p with clusters of lysosomes and late endosomes. HeLa cells were transiently transfected with HA–hVam6p and processed for immunoelectron microscopy. Ultra-thin frozen sections were labeled with antibodies to HA (to detect hVam6p) and either endogenous lamp-2 (A) or endogenous CI-MPR (B). Bound antibodies were detected using conjugated protein A–gold. Arrows indicate the localization of hVam6p to a halo around the membranes of lysosomes and late endosomes (A and B, 10-nm gold particles), whereas arrowheads indicate the localization of lamp-2 (A, 15-nm gold particles) or CI-MPR (B, 15-nm gold particles), and the asterisk marks a structure labeled only for hVam6p. G, Golgi. Bars, 0.2 μm.
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fig3: Association of hVam6p with clusters of lysosomes and late endosomes. HeLa cells were transiently transfected with HA–hVam6p and processed for immunoelectron microscopy. Ultra-thin frozen sections were labeled with antibodies to HA (to detect hVam6p) and either endogenous lamp-2 (A) or endogenous CI-MPR (B). Bound antibodies were detected using conjugated protein A–gold. Arrows indicate the localization of hVam6p to a halo around the membranes of lysosomes and late endosomes (A and B, 10-nm gold particles), whereas arrowheads indicate the localization of lamp-2 (A, 15-nm gold particles) or CI-MPR (B, 15-nm gold particles), and the asterisk marks a structure labeled only for hVam6p. G, Golgi. Bars, 0.2 μm.
Mentions: The localization of hVam6p in transfected HeLa cells was further examined by immunoelectron microscopy of ultrathin frozen sections (Fig. 3) . hVam6p was found to be associated with an electron dense halo surrounding 0.2–0.6 μm vesicles that were part of large clusters (Fig. 3 A, 10-nm gold particles, arrows). All of these vesicles were also labeled for the lysosomal membrane protein, lamp-2 (Fig. 3 A, 15-nm gold particles, arrowheads). Although many hVam6p-coated vesicles did not contain cation-independent mannose 6-phosphate receptor (CI-MPR) (Fig 3 B, *), others contained both hVam6p (Fig. 3 B, 10-nm gold particles, arrows) and CI-MPR (Fig. 3 B, 15-nm gold particles, arrowhead), and some had the appearance of multivesicular bodies. Together, these observations suggest that hVam6p associates with and induces clustering of lysosomes and late endosomes.

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