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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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Ultrastructural analysis of hVam6p-induced lysosomal structures labeled with internalized HRP. Untransfected (A) or hVam6p-transfected (B–E) HeLa cells were subjected to a continuous 4 h fluid phase uptake of HRP 24 h after transfection. (A) In control cells, HRP-containing lysosomes and endosomes were small (0.2–0.6 μm) and distributed throughout cell. (B) In hVam6p-transfected cells, clusters of multivesicular HRP-positive vesicles (enlarged in C) and large (2–3 μm) vacuoles situated next to the nucleus were visible. Some of these large vacuoles contained HRP (enlarged in D), whereas others appeared to have smaller HRP-positive vesicles docking onto their membrane (enlarged in E). N, Nucleus. Bars: (A and B) 2 μm; (C–E) 0.4 μm.
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fig5: Ultrastructural analysis of hVam6p-induced lysosomal structures labeled with internalized HRP. Untransfected (A) or hVam6p-transfected (B–E) HeLa cells were subjected to a continuous 4 h fluid phase uptake of HRP 24 h after transfection. (A) In control cells, HRP-containing lysosomes and endosomes were small (0.2–0.6 μm) and distributed throughout cell. (B) In hVam6p-transfected cells, clusters of multivesicular HRP-positive vesicles (enlarged in C) and large (2–3 μm) vacuoles situated next to the nucleus were visible. Some of these large vacuoles contained HRP (enlarged in D), whereas others appeared to have smaller HRP-positive vesicles docking onto their membrane (enlarged in E). N, Nucleus. Bars: (A and B) 2 μm; (C–E) 0.4 μm.

Mentions: We took advantage of the ability to load the hVam6p-induced lysosomal structures with fluid phase endocytic markers to analyze their ultrastructure in more detail. Untransfected or hVam6p-transfected HeLa cells were allowed to internalize HRP for 4 h. After standard fixation, diaminobenzidine development for HRP visualization, and resin embedding, cell sections were analyzed by electron microscopy. As expected, untransfected cells displayed an array of 0.2–0.6-μm HRP-positive vesicles scattered throughout the cytoplasm, most of which likely corresponded to late endosomes and lysosomes because of the long period of internalization (Fig. 5 A). hVam6p-transfected HeLa cells, on the other hand, contained at least three types of abnormal structures. The first type consisted of large clusters of HRP-positive 0.2–0.6 μM vesicles, most of which contained intraluminal vesicles or other membranous inclusions (Fig. 5, B and C), similar to those seen on ultrathin cryosections (Fig. 3). The second type of abnormal structures were large (2–3 μm) vacuoles (Fig. 5, B and D). Some of these vacuoles seemed empty, displaying the appearance of swollen vacuoles. Others had variable amounts of HRP-positive materials, including 0.2–0.6 μM vesicles, within their interior (Fig. 5 D). The third type was a combination of the former two in that clusters of 0.2–0.6-μM HRP-positive vesicles were docked onto the membranes of the large vacuoles (Fig. 5, B and E). Serial sectioning (not shown) revealed that these three types of structures were situated next to the nucleus, often nestled between nuclear lobes. These analyses suggested a possible series of events induced by hVam6p, in which late endosomes and lysosomes first cluster together and then undergo fusion to generate large vacuoles.


Human Vam6p promotes lysosome clustering and fusion in vivo.

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

Ultrastructural analysis of hVam6p-induced lysosomal structures labeled with internalized HRP. Untransfected (A) or hVam6p-transfected (B–E) HeLa cells were subjected to a continuous 4 h fluid phase uptake of HRP 24 h after transfection. (A) In control cells, HRP-containing lysosomes and endosomes were small (0.2–0.6 μm) and distributed throughout cell. (B) In hVam6p-transfected cells, clusters of multivesicular HRP-positive vesicles (enlarged in C) and large (2–3 μm) vacuoles situated next to the nucleus were visible. Some of these large vacuoles contained HRP (enlarged in D), whereas others appeared to have smaller HRP-positive vesicles docking onto their membrane (enlarged in E). N, Nucleus. Bars: (A and B) 2 μm; (C–E) 0.4 μm.
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Related In: Results  -  Collection

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fig5: Ultrastructural analysis of hVam6p-induced lysosomal structures labeled with internalized HRP. Untransfected (A) or hVam6p-transfected (B–E) HeLa cells were subjected to a continuous 4 h fluid phase uptake of HRP 24 h after transfection. (A) In control cells, HRP-containing lysosomes and endosomes were small (0.2–0.6 μm) and distributed throughout cell. (B) In hVam6p-transfected cells, clusters of multivesicular HRP-positive vesicles (enlarged in C) and large (2–3 μm) vacuoles situated next to the nucleus were visible. Some of these large vacuoles contained HRP (enlarged in D), whereas others appeared to have smaller HRP-positive vesicles docking onto their membrane (enlarged in E). N, Nucleus. Bars: (A and B) 2 μm; (C–E) 0.4 μm.
Mentions: We took advantage of the ability to load the hVam6p-induced lysosomal structures with fluid phase endocytic markers to analyze their ultrastructure in more detail. Untransfected or hVam6p-transfected HeLa cells were allowed to internalize HRP for 4 h. After standard fixation, diaminobenzidine development for HRP visualization, and resin embedding, cell sections were analyzed by electron microscopy. As expected, untransfected cells displayed an array of 0.2–0.6-μm HRP-positive vesicles scattered throughout the cytoplasm, most of which likely corresponded to late endosomes and lysosomes because of the long period of internalization (Fig. 5 A). hVam6p-transfected HeLa cells, on the other hand, contained at least three types of abnormal structures. The first type consisted of large clusters of HRP-positive 0.2–0.6 μM vesicles, most of which contained intraluminal vesicles or other membranous inclusions (Fig. 5, B and C), similar to those seen on ultrathin cryosections (Fig. 3). The second type of abnormal structures were large (2–3 μm) vacuoles (Fig. 5, B and D). Some of these vacuoles seemed empty, displaying the appearance of swollen vacuoles. Others had variable amounts of HRP-positive materials, including 0.2–0.6 μM vesicles, within their interior (Fig. 5 D). The third type was a combination of the former two in that clusters of 0.2–0.6-μM HRP-positive vesicles were docked onto the membranes of the large vacuoles (Fig. 5, B and E). Serial sectioning (not shown) revealed that these three types of structures were situated next to the nucleus, often nestled between nuclear lobes. These analyses suggested a possible series of events induced by hVam6p, in which late endosomes and lysosomes first cluster together and then undergo fusion to generate large vacuoles.

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