Limits...
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.

Show MeSH
hVam6p affects lysosomal morphology independently of Rab7 nucleotide cycling. HeLa cells were transfected with either GFP–Rab7 (A and B, control) or dominant-negative GFP–Rab7 T22N (E and F, control), or were cotransfected with Myc–hVam6p together with either GFP–Rab7 (C and D) or GFP–Rab7 T22N (G and H). After 24 h, the cells were fixed-permeabilized and incubated with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–mouse IgG (B, D, F, and H), and expression of GFP–Rab7 proteins visualized by their intrinsic fluorescence (A, C, E, and G). Arrowheads depict the coalescence of both GFP–Rab7 (C) and lamp-1 (D) to a large juxtanuclear conglomerate. Small arrows (F) mark the localization of dispersed lysosomes in cells transfected with Rab7 T22N. Large arrow (H) denotes a giant juxtanuclear lysosome conglomerate in a cell overexpressing GFP–Rab7 T22N and Myc–hVam6p. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196876&req=5

fig7: hVam6p affects lysosomal morphology independently of Rab7 nucleotide cycling. HeLa cells were transfected with either GFP–Rab7 (A and B, control) or dominant-negative GFP–Rab7 T22N (E and F, control), or were cotransfected with Myc–hVam6p together with either GFP–Rab7 (C and D) or GFP–Rab7 T22N (G and H). After 24 h, the cells were fixed-permeabilized and incubated with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–mouse IgG (B, D, F, and H), and expression of GFP–Rab7 proteins visualized by their intrinsic fluorescence (A, C, E, and G). Arrowheads depict the coalescence of both GFP–Rab7 (C) and lamp-1 (D) to a large juxtanuclear conglomerate. Small arrows (F) mark the localization of dispersed lysosomes in cells transfected with Rab7 T22N. Large arrow (H) denotes a giant juxtanuclear lysosome conglomerate in a cell overexpressing GFP–Rab7 T22N and Myc–hVam6p. Bar, 10 μm.

Mentions: The effects of hVam6p on lysosomes are reminiscent of those elicited by a constitutively active Rab7 Q67L mutant (Bucci et al., 2000). To determine whether Rab7 nucleotide cycling is necessary for the formation of hVam6p-induced lysosome clusters, we transfected cells with wild-type GFP–Rab7, constitutively activated GFP–Rab7 Q67L, or dominant-negative GFP–Rab7 T22N, each alone or together with hVam6p. Overexpression of wild type GFP–Rab7 did not affect the distribution of endogenous lamp-1 (Fig. 7, A and B) as previously reported (Bucci et al., 2000). However, in cells transfected with GFP–Rab7 and hVam6p, both GFP–Rab7 and lamp-1 were found in large juxtanuclear clusters (Fig. 7, C and D, arrowheads). Since GFP–Rab7 Q67L induces an effect on lysosomes (Bucci et al., 2000) that resembles that of hVam6p, we were unable to discern any additional effect in cells expressing both of these proteins (data not shown). However, GFP–Rab7 T22N, which by itself causes dispersal of lysosomes from the juxtanuclear region to the periphery (Bucci et al., 2000; and Fig. 7, E and F, small arrows) did not block the coalescence of lysosomes induced by hVam6p (Fig. 7, G and H, large arrow). These findings imply that hVam6p exerts its affects either downstream of or in parallel to Rab7.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

hVam6p affects lysosomal morphology independently of Rab7 nucleotide cycling. HeLa cells were transfected with either GFP–Rab7 (A and B, control) or dominant-negative GFP–Rab7 T22N (E and F, control), or were cotransfected with Myc–hVam6p together with either GFP–Rab7 (C and D) or GFP–Rab7 T22N (G and H). After 24 h, the cells were fixed-permeabilized and incubated with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–mouse IgG (B, D, F, and H), and expression of GFP–Rab7 proteins visualized by their intrinsic fluorescence (A, C, E, and G). Arrowheads depict the coalescence of both GFP–Rab7 (C) and lamp-1 (D) to a large juxtanuclear conglomerate. Small arrows (F) mark the localization of dispersed lysosomes in cells transfected with Rab7 T22N. Large arrow (H) denotes a giant juxtanuclear lysosome conglomerate in a cell overexpressing GFP–Rab7 T22N and Myc–hVam6p. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196876&req=5

fig7: hVam6p affects lysosomal morphology independently of Rab7 nucleotide cycling. HeLa cells were transfected with either GFP–Rab7 (A and B, control) or dominant-negative GFP–Rab7 T22N (E and F, control), or were cotransfected with Myc–hVam6p together with either GFP–Rab7 (C and D) or GFP–Rab7 T22N (G and H). After 24 h, the cells were fixed-permeabilized and incubated with mouse monoclonal antibodies to lamp-1. Bound antibodies were revealed by Cy3-conjugated donkey anti–mouse IgG (B, D, F, and H), and expression of GFP–Rab7 proteins visualized by their intrinsic fluorescence (A, C, E, and G). Arrowheads depict the coalescence of both GFP–Rab7 (C) and lamp-1 (D) to a large juxtanuclear conglomerate. Small arrows (F) mark the localization of dispersed lysosomes in cells transfected with Rab7 T22N. Large arrow (H) denotes a giant juxtanuclear lysosome conglomerate in a cell overexpressing GFP–Rab7 T22N and Myc–hVam6p. Bar, 10 μm.
Mentions: The effects of hVam6p on lysosomes are reminiscent of those elicited by a constitutively active Rab7 Q67L mutant (Bucci et al., 2000). To determine whether Rab7 nucleotide cycling is necessary for the formation of hVam6p-induced lysosome clusters, we transfected cells with wild-type GFP–Rab7, constitutively activated GFP–Rab7 Q67L, or dominant-negative GFP–Rab7 T22N, each alone or together with hVam6p. Overexpression of wild type GFP–Rab7 did not affect the distribution of endogenous lamp-1 (Fig. 7, A and B) as previously reported (Bucci et al., 2000). However, in cells transfected with GFP–Rab7 and hVam6p, both GFP–Rab7 and lamp-1 were found in large juxtanuclear clusters (Fig. 7, C and D, arrowheads). Since GFP–Rab7 Q67L induces an effect on lysosomes (Bucci et al., 2000) that resembles that of hVam6p, we were unable to discern any additional effect in cells expressing both of these proteins (data not shown). However, GFP–Rab7 T22N, which by itself causes dispersal of lysosomes from the juxtanuclear region to the periphery (Bucci et al., 2000; and Fig. 7, E and F, small arrows) did not block the coalescence of lysosomes induced by hVam6p (Fig. 7, G and H, large arrow). These findings imply that hVam6p exerts its affects either downstream of or in parallel to Rab7.

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