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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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Functional characterization of hVam6p-induced lysosomal clusters. (A–F) Live HeLa cells transiently transfected with Myc–hVam6p were incubated with LysoTracker red (A–C) or rhodamine–dextran (D–F) for 1 h, and then fixed-permeabilized. Cells were incubated with a mouse monoclonal antibody to Myc, followed by Alexa-488–conjugated donkey antibody to mouse IgG (B and E, green channel). The LysoTracker red and rhodamine–dextran are visible in the red channel (A and D). Arrows denote the accumulation of acidotropic (A) and fluid phase (D) markers within clustered lysosomes in hVam6p-transfected cells. Bar, 10 μm. (G) HeLa cells were cotransfected with either Tac-DKQTLL and Myc–hVam6p, or Tac-DKQTLL and a nonmyristylated Arf6 control. After 24 h, cotransfection efficiency was monitored by indirect immunofluorescence (as described in the legend to Fig. 2), and cells were pulsed for 30 min either by metabolic labeling (top) or cell surface biotinylation (bottom). The cells were then chased for the time points indicated, harvested, lysed, subjected to immunoprecipitation analysis using antibodies directed against Tac, and resolved on 4–20% SDS-PAGE. Samples from metabolically labeled and biotinylated cells were visualized by autoradiography and blotting with streptavidin–HRP, respectively.
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fig4: Functional characterization of hVam6p-induced lysosomal clusters. (A–F) Live HeLa cells transiently transfected with Myc–hVam6p were incubated with LysoTracker red (A–C) or rhodamine–dextran (D–F) for 1 h, and then fixed-permeabilized. Cells were incubated with a mouse monoclonal antibody to Myc, followed by Alexa-488–conjugated donkey antibody to mouse IgG (B and E, green channel). The LysoTracker red and rhodamine–dextran are visible in the red channel (A and D). Arrows denote the accumulation of acidotropic (A) and fluid phase (D) markers within clustered lysosomes in hVam6p-transfected cells. Bar, 10 μm. (G) HeLa cells were cotransfected with either Tac-DKQTLL and Myc–hVam6p, or Tac-DKQTLL and a nonmyristylated Arf6 control. After 24 h, cotransfection efficiency was monitored by indirect immunofluorescence (as described in the legend to Fig. 2), and cells were pulsed for 30 min either by metabolic labeling (top) or cell surface biotinylation (bottom). The cells were then chased for the time points indicated, harvested, lysed, subjected to immunoprecipitation analysis using antibodies directed against Tac, and resolved on 4–20% SDS-PAGE. Samples from metabolically labeled and biotinylated cells were visualized by autoradiography and blotting with streptavidin–HRP, respectively.

Mentions: The hVam6p-induced lysosomal structures could be labeled with the acidotropic fluorescent probe, LysoTracker red, indicating that they were acidic (Fig. 4 , A–C, arrow). To determine if they were also accessible to fluid-phase markers, HeLa cells expressing Myc–hVam6p were allowed to internalize the fluid phase marker rhodamine–dextran for various time periods. Cells were then fixed permeabilized cells and subjected to indirect immunofluorescence to detect hVam6p-expressing cells. At time points ranging from 15 min (not shown) to 1 h (Fig. 4, D–F), rhodamine–dextran was found to be internalized equally well into typical lysosomes in untransfected cells and lysosomal clusters in hVam6p-transfected cells (Fig. 4 D, arrows), suggesting no major change in accessibility to fluid phase markers upon expression of hVam6p.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

Functional characterization of hVam6p-induced lysosomal clusters. (A–F) Live HeLa cells transiently transfected with Myc–hVam6p were incubated with LysoTracker red (A–C) or rhodamine–dextran (D–F) for 1 h, and then fixed-permeabilized. Cells were incubated with a mouse monoclonal antibody to Myc, followed by Alexa-488–conjugated donkey antibody to mouse IgG (B and E, green channel). The LysoTracker red and rhodamine–dextran are visible in the red channel (A and D). Arrows denote the accumulation of acidotropic (A) and fluid phase (D) markers within clustered lysosomes in hVam6p-transfected cells. Bar, 10 μm. (G) HeLa cells were cotransfected with either Tac-DKQTLL and Myc–hVam6p, or Tac-DKQTLL and a nonmyristylated Arf6 control. After 24 h, cotransfection efficiency was monitored by indirect immunofluorescence (as described in the legend to Fig. 2), and cells were pulsed for 30 min either by metabolic labeling (top) or cell surface biotinylation (bottom). The cells were then chased for the time points indicated, harvested, lysed, subjected to immunoprecipitation analysis using antibodies directed against Tac, and resolved on 4–20% SDS-PAGE. Samples from metabolically labeled and biotinylated cells were visualized by autoradiography and blotting with streptavidin–HRP, respectively.
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fig4: Functional characterization of hVam6p-induced lysosomal clusters. (A–F) Live HeLa cells transiently transfected with Myc–hVam6p were incubated with LysoTracker red (A–C) or rhodamine–dextran (D–F) for 1 h, and then fixed-permeabilized. Cells were incubated with a mouse monoclonal antibody to Myc, followed by Alexa-488–conjugated donkey antibody to mouse IgG (B and E, green channel). The LysoTracker red and rhodamine–dextran are visible in the red channel (A and D). Arrows denote the accumulation of acidotropic (A) and fluid phase (D) markers within clustered lysosomes in hVam6p-transfected cells. Bar, 10 μm. (G) HeLa cells were cotransfected with either Tac-DKQTLL and Myc–hVam6p, or Tac-DKQTLL and a nonmyristylated Arf6 control. After 24 h, cotransfection efficiency was monitored by indirect immunofluorescence (as described in the legend to Fig. 2), and cells were pulsed for 30 min either by metabolic labeling (top) or cell surface biotinylation (bottom). The cells were then chased for the time points indicated, harvested, lysed, subjected to immunoprecipitation analysis using antibodies directed against Tac, and resolved on 4–20% SDS-PAGE. Samples from metabolically labeled and biotinylated cells were visualized by autoradiography and blotting with streptavidin–HRP, respectively.
Mentions: The hVam6p-induced lysosomal structures could be labeled with the acidotropic fluorescent probe, LysoTracker red, indicating that they were acidic (Fig. 4 , A–C, arrow). To determine if they were also accessible to fluid-phase markers, HeLa cells expressing Myc–hVam6p were allowed to internalize the fluid phase marker rhodamine–dextran for various time periods. Cells were then fixed permeabilized cells and subjected to indirect immunofluorescence to detect hVam6p-expressing cells. At time points ranging from 15 min (not shown) to 1 h (Fig. 4, D–F), rhodamine–dextran was found to be internalized equally well into typical lysosomes in untransfected cells and lysosomal clusters in hVam6p-transfected cells (Fig. 4 D, arrows), suggesting no major change in accessibility to fluid phase markers upon expression of hVam6p.

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