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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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Delineation of functional domains in hVam6p. (A) Schematic representation of full-length hVam6p and various deletion constructs used in these experiments. (B) HeLa cells were cotranfected with plasmids encoding Myc– and HA–hVam6p, or hVam6p deletion mutants, or a control epitope-tagged protein (HA–JNK1), as indicated. After 18 h, cells were labeled for 8 h with [35S]methionine, detergent extracted, and subjected to immunoprecipitation–recapture with the antibodies indicated. (C-N) HeLa cells were transfected with plasmids encoding Myc– or HA–Vam6p full-length or deletion constructs. After 24 h, fixed-permeabilized cells were coincubated with rabbit polyclonal antibodies to either the Myc or HA epitopes together with mouse monoclonal antibody to lamp-1. Bound antibodies were revealed by Alexa-488–conjugated donkey anti–mouse antibody (green channel) and Cy3-conjugated donkey anti–rabbit IgG (red channel). The third panel in each row was generated by merging of the images in the red and green channels. Arrows mark the coalescence of lysosomes into juxtanuclear regions. Bar, 10 μm.
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fig9: Delineation of functional domains in hVam6p. (A) Schematic representation of full-length hVam6p and various deletion constructs used in these experiments. (B) HeLa cells were cotranfected with plasmids encoding Myc– and HA–hVam6p, or hVam6p deletion mutants, or a control epitope-tagged protein (HA–JNK1), as indicated. After 18 h, cells were labeled for 8 h with [35S]methionine, detergent extracted, and subjected to immunoprecipitation–recapture with the antibodies indicated. (C-N) HeLa cells were transfected with plasmids encoding Myc– or HA–Vam6p full-length or deletion constructs. After 24 h, fixed-permeabilized cells were coincubated with rabbit polyclonal antibodies to either the Myc or HA epitopes together with mouse monoclonal antibody to lamp-1. Bound antibodies were revealed by Alexa-488–conjugated donkey anti–mouse antibody (green channel) and Cy3-conjugated donkey anti–rabbit IgG (red channel). The third panel in each row was generated by merging of the images in the red and green channels. Arrows mark the coalescence of lysosomes into juxtanuclear regions. Bar, 10 μm.

Mentions: To further assess the self-association of hVam6p in vivo, HeLa cells were cotransfected with Myc–hVam6p and HA–hVam6p (Fig. 9 B). After metabolic labeling, the cells were lysed and subjected to immunoprecipitation–recapture analysis. Sequential immunoprecipitation with antibodies to Myc and to HA demonstrated that the two epitope-tagged hVam6p proteins interacted in vivo (Fig. 9 B). The amount of total HA-tagged hVam6p coprecipitated with antibody to Myc was 10–20% in several experiments. Although this percentage may seem low, several combinations of epitope-tagged proteins can be formed, namely Myc–Myc, HA–HA, and Myc–HA. In addition, the immunoprecipitation–recapture procedure is not quantitative because of the presence of some SDS in the recapture step. Accordingly, the level of Myc–HA recaptured hVam6p probably represents a significant fraction of the total hVam6p. The coprecipitation was specific, as Myc–hVam6p did not coprecipitate with HA-tagged JNK1 (Fig. 9 B). These results support the observations from the sucrose gradient analyses, indicating that hVam6p is a homooligomer.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

Delineation of functional domains in hVam6p. (A) Schematic representation of full-length hVam6p and various deletion constructs used in these experiments. (B) HeLa cells were cotranfected with plasmids encoding Myc– and HA–hVam6p, or hVam6p deletion mutants, or a control epitope-tagged protein (HA–JNK1), as indicated. After 18 h, cells were labeled for 8 h with [35S]methionine, detergent extracted, and subjected to immunoprecipitation–recapture with the antibodies indicated. (C-N) HeLa cells were transfected with plasmids encoding Myc– or HA–Vam6p full-length or deletion constructs. After 24 h, fixed-permeabilized cells were coincubated with rabbit polyclonal antibodies to either the Myc or HA epitopes together with mouse monoclonal antibody to lamp-1. Bound antibodies were revealed by Alexa-488–conjugated donkey anti–mouse antibody (green channel) and Cy3-conjugated donkey anti–rabbit IgG (red channel). The third panel in each row was generated by merging of the images in the red and green channels. Arrows mark the coalescence of lysosomes into juxtanuclear regions. Bar, 10 μm.
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Related In: Results  -  Collection

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fig9: Delineation of functional domains in hVam6p. (A) Schematic representation of full-length hVam6p and various deletion constructs used in these experiments. (B) HeLa cells were cotranfected with plasmids encoding Myc– and HA–hVam6p, or hVam6p deletion mutants, or a control epitope-tagged protein (HA–JNK1), as indicated. After 18 h, cells were labeled for 8 h with [35S]methionine, detergent extracted, and subjected to immunoprecipitation–recapture with the antibodies indicated. (C-N) HeLa cells were transfected with plasmids encoding Myc– or HA–Vam6p full-length or deletion constructs. After 24 h, fixed-permeabilized cells were coincubated with rabbit polyclonal antibodies to either the Myc or HA epitopes together with mouse monoclonal antibody to lamp-1. Bound antibodies were revealed by Alexa-488–conjugated donkey anti–mouse antibody (green channel) and Cy3-conjugated donkey anti–rabbit IgG (red channel). The third panel in each row was generated by merging of the images in the red and green channels. Arrows mark the coalescence of lysosomes into juxtanuclear regions. Bar, 10 μm.
Mentions: To further assess the self-association of hVam6p in vivo, HeLa cells were cotransfected with Myc–hVam6p and HA–hVam6p (Fig. 9 B). After metabolic labeling, the cells were lysed and subjected to immunoprecipitation–recapture analysis. Sequential immunoprecipitation with antibodies to Myc and to HA demonstrated that the two epitope-tagged hVam6p proteins interacted in vivo (Fig. 9 B). The amount of total HA-tagged hVam6p coprecipitated with antibody to Myc was 10–20% in several experiments. Although this percentage may seem low, several combinations of epitope-tagged proteins can be formed, namely Myc–Myc, HA–HA, and Myc–HA. In addition, the immunoprecipitation–recapture procedure is not quantitative because of the presence of some SDS in the recapture step. Accordingly, the level of Myc–HA recaptured hVam6p probably represents a significant fraction of the total hVam6p. The coprecipitation was specific, as Myc–hVam6p did not coprecipitate with HA-tagged JNK1 (Fig. 9 B). These results support the observations from the sucrose gradient analyses, indicating that hVam6p is a homooligomer.

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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