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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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Overexpression of hVam6p induces coalescence of lysosomal vesicles. (A–O) HeLa cells were transiently transfected with a plasmid encoding Myc–hVam6p. Cells were fixed, permeabilized, and incubated with rabbit polyclonal antibodies to the Myc epitope together with mouse monoclonal antibodies to lamp-1 (A–C), lamp-2 (D–F), CD63 (G–I), or transferrin receptor (M–O), or rabbit polyclonal antibody to cathepsin D (J–L). Arrows (A, D, G, and J) indicate the coalescence of lysosomes into large juxtanuclear structures. Arrowhead (M) indicates a transfected cell. (P–R) HA–hVam6p-transfected HeLa cells were extracted for 1 min with 0.05% (wt/vol) saponin before fixation. Cells were then fixed and incubated with rabbit polyclonal antibodies to the HA epitope, together with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–rabbit IgG (red channel) and Alexa-488–conjugated donkey anti–mouse antibody (green channel). Bar, 10 μm.
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fig2: Overexpression of hVam6p induces coalescence of lysosomal vesicles. (A–O) HeLa cells were transiently transfected with a plasmid encoding Myc–hVam6p. Cells were fixed, permeabilized, and incubated with rabbit polyclonal antibodies to the Myc epitope together with mouse monoclonal antibodies to lamp-1 (A–C), lamp-2 (D–F), CD63 (G–I), or transferrin receptor (M–O), or rabbit polyclonal antibody to cathepsin D (J–L). Arrows (A, D, G, and J) indicate the coalescence of lysosomes into large juxtanuclear structures. Arrowhead (M) indicates a transfected cell. (P–R) HA–hVam6p-transfected HeLa cells were extracted for 1 min with 0.05% (wt/vol) saponin before fixation. Cells were then fixed and incubated with rabbit polyclonal antibodies to the HA epitope, together with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–rabbit IgG (red channel) and Alexa-488–conjugated donkey anti–mouse antibody (green channel). Bar, 10 μm.

Mentions: S. cerevisiae Vam6p has been implicated in tethering and/or docking events that precede homotypic vacuole fusion (Eitzen et al., 2000; Price et al., 2000a,b; Seals et al., 2000; Wurmser et al., 2000). To assess whether hVam6p could play a similar role in lysosome tethering/docking, we examined the effects of overexpressing Myc epitope–tagged hVam6p (Myc–hVam6p) by transient transfection into HeLa cells. Fixed-permeabilized cells were analyzed by indirect immunofluorescence microscopy after double labeling with antibodies to the Myc epitope and to the lysosomal integral membrane proteins, lamp-1 (Fig. 2 , A–C), lamp-2 (Fig. 2, D–F), and CD63 (Fig. 2, G–I). Untransfected cells exhibited the characteristic distribution of lysosomes, which were more concentrated in the juxtanuclear area of the cytoplasm but also extended toward the cell periphery (Fig. 2, A–I). In contrast, cells overexpressing hVam6p displayed a striking coalescence of lysosomes into a few large juxtanuclear structures (Fig. 2, A–I, arrows) with concomitant loss of peripheral lysosomes. These large structures were observed in virtually all hVam6p-overexpressing cells and contained all three lysosomal membrane proteins tested, as well as the lysosomal luminal hydrolase, cathepsin D (Fig. 2, J–L, arrows). The distributions of the early endosomal marker, transferrin receptor, in cells overexpressing hVam6p (Fig. 2, M–O, arrowhead), as well as various other endosomal, TGN, and Golgi markers (i.e., EEA1, AP-1, and the 58-kD Golgi protein; data not shown), were not affected. These observations suggested that the morphological alterations induced by overexpression of hVam6p were specific to organelles containing lysosomal membrane and luminal proteins.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

Overexpression of hVam6p induces coalescence of lysosomal vesicles. (A–O) HeLa cells were transiently transfected with a plasmid encoding Myc–hVam6p. Cells were fixed, permeabilized, and incubated with rabbit polyclonal antibodies to the Myc epitope together with mouse monoclonal antibodies to lamp-1 (A–C), lamp-2 (D–F), CD63 (G–I), or transferrin receptor (M–O), or rabbit polyclonal antibody to cathepsin D (J–L). Arrows (A, D, G, and J) indicate the coalescence of lysosomes into large juxtanuclear structures. Arrowhead (M) indicates a transfected cell. (P–R) HA–hVam6p-transfected HeLa cells were extracted for 1 min with 0.05% (wt/vol) saponin before fixation. Cells were then fixed and incubated with rabbit polyclonal antibodies to the HA epitope, together with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–rabbit IgG (red channel) and Alexa-488–conjugated donkey anti–mouse antibody (green channel). Bar, 10 μm.
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Related In: Results  -  Collection

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fig2: Overexpression of hVam6p induces coalescence of lysosomal vesicles. (A–O) HeLa cells were transiently transfected with a plasmid encoding Myc–hVam6p. Cells were fixed, permeabilized, and incubated with rabbit polyclonal antibodies to the Myc epitope together with mouse monoclonal antibodies to lamp-1 (A–C), lamp-2 (D–F), CD63 (G–I), or transferrin receptor (M–O), or rabbit polyclonal antibody to cathepsin D (J–L). Arrows (A, D, G, and J) indicate the coalescence of lysosomes into large juxtanuclear structures. Arrowhead (M) indicates a transfected cell. (P–R) HA–hVam6p-transfected HeLa cells were extracted for 1 min with 0.05% (wt/vol) saponin before fixation. Cells were then fixed and incubated with rabbit polyclonal antibodies to the HA epitope, together with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–rabbit IgG (red channel) and Alexa-488–conjugated donkey anti–mouse antibody (green channel). Bar, 10 μm.
Mentions: S. cerevisiae Vam6p has been implicated in tethering and/or docking events that precede homotypic vacuole fusion (Eitzen et al., 2000; Price et al., 2000a,b; Seals et al., 2000; Wurmser et al., 2000). To assess whether hVam6p could play a similar role in lysosome tethering/docking, we examined the effects of overexpressing Myc epitope–tagged hVam6p (Myc–hVam6p) by transient transfection into HeLa cells. Fixed-permeabilized cells were analyzed by indirect immunofluorescence microscopy after double labeling with antibodies to the Myc epitope and to the lysosomal integral membrane proteins, lamp-1 (Fig. 2 , A–C), lamp-2 (Fig. 2, D–F), and CD63 (Fig. 2, G–I). Untransfected cells exhibited the characteristic distribution of lysosomes, which were more concentrated in the juxtanuclear area of the cytoplasm but also extended toward the cell periphery (Fig. 2, A–I). In contrast, cells overexpressing hVam6p displayed a striking coalescence of lysosomes into a few large juxtanuclear structures (Fig. 2, A–I, arrows) with concomitant loss of peripheral lysosomes. These large structures were observed in virtually all hVam6p-overexpressing cells and contained all three lysosomal membrane proteins tested, as well as the lysosomal luminal hydrolase, cathepsin D (Fig. 2, J–L, arrows). The distributions of the early endosomal marker, transferrin receptor, in cells overexpressing hVam6p (Fig. 2, M–O, arrowhead), as well as various other endosomal, TGN, and Golgi markers (i.e., EEA1, AP-1, and the 58-kD Golgi protein; data not shown), were not affected. These observations suggested that the morphological alterations induced by overexpression of hVam6p were specific to organelles containing lysosomal membrane and luminal proteins.

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