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Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel.

Bayer MJ, Reese C, Buhler S, Peters C, Mayer A - J. Cell Biol. (2003)

Bottom Line: Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse.The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release.The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release.

View Article: PubMed Central - PubMed

Affiliation: Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tübingen, Germany.

ABSTRACT
Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.

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Vacuolar structure in V-ATPase mutants. (A) Yeast cells (BY4742) containing the indicated deletions of different V-ATPase subunits were grown logarithmically in liquid YPD medium, stained with FM4–64, and analyzed by fluorescence microscopy as described in Materials and methods. (B) Quantitation of vacuole morphology. The number of vacuolar vesicles (stained by FM4–64) per cell was determined for the strains shown in A. For each experiment, 200 cells per strain were analyzed and grouped into the indicated categories. Two experiments were averaged. Bar, 5 μm.
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fig1: Vacuolar structure in V-ATPase mutants. (A) Yeast cells (BY4742) containing the indicated deletions of different V-ATPase subunits were grown logarithmically in liquid YPD medium, stained with FM4–64, and analyzed by fluorescence microscopy as described in Materials and methods. (B) Quantitation of vacuole morphology. The number of vacuolar vesicles (stained by FM4–64) per cell was determined for the strains shown in A. For each experiment, 200 cells per strain were analyzed and grouped into the indicated categories. Two experiments were averaged. Bar, 5 μm.

Mentions: Absence of the vma phenotype makes vph1 mutants suitable for probing a role of V0 in vacuolar fusion in vivo. Mutation of fusion-relevant components frequently results in a fragmentation of vacuoles into multiple small vesicles in vivo (Raymond et al., 1992; Wada et al., 1992b). In some mutants, fragmentation does not occur, for example, after mutation of the v-SNARE Nyv1, Vtc1, or Vtc4 (Nichols et al., 1997; Muller et al., 2002). We assayed the in vivo effects of V-ATPase mutations by light microscopy after staining the vacuolar membranes with the red fluorescent vital dye FM4–64. Mutants lacking the vacuolar V0 subunit Vph1p showed numerous small vacuolar fragments which formed clusters (Fig. 1). This phenotype was observed with high frequency and resembles that of mutants in other genes with a function in the postdocking phase of vacuole fusion, such as Vtc3, Glc7 (protein phosphatase 1), calmodulin, or Vac8 (Peters and Mayer, 1998; Peters et al., 1999, 2001; Wang et al., 2001b; Muller et al., 2002). In contrast, mutation of Stv1, the Golgi/endosomal isoform of Vph1, did not result in vacuolar fragmentation (Fig. 1), nor did deletion of the V1 subunit Vma1p (unpublished data; with the strong caveat that this last mutant has the severe vma phenotype). Our results differ from those published in another study (Perzov et al., 2002) that reported vacuolar fragmentation for Δstv1 cells but not for Δvph1 cells. Due to this discrepancy, we generated Δvph1 mutants in three independent strain backgrounds and consistently observed vacuolar fragmentation. Currently, we have no explanation for this difference. However, our results are strongly corroborated by an unbiased microscopic screen for deletion mutants showing vacuolar fragmentation (Seeley et al., 2002). This genome wide screen identified mutants in four out of five Vo subunits as defective in vacuolar morphology, reporting a particularly strong phenotype for Δvph1 but none for Δstv1 or Δvma1.


Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel.

Bayer MJ, Reese C, Buhler S, Peters C, Mayer A - J. Cell Biol. (2003)

Vacuolar structure in V-ATPase mutants. (A) Yeast cells (BY4742) containing the indicated deletions of different V-ATPase subunits were grown logarithmically in liquid YPD medium, stained with FM4–64, and analyzed by fluorescence microscopy as described in Materials and methods. (B) Quantitation of vacuole morphology. The number of vacuolar vesicles (stained by FM4–64) per cell was determined for the strains shown in A. For each experiment, 200 cells per strain were analyzed and grouped into the indicated categories. Two experiments were averaged. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172786&req=5

fig1: Vacuolar structure in V-ATPase mutants. (A) Yeast cells (BY4742) containing the indicated deletions of different V-ATPase subunits were grown logarithmically in liquid YPD medium, stained with FM4–64, and analyzed by fluorescence microscopy as described in Materials and methods. (B) Quantitation of vacuole morphology. The number of vacuolar vesicles (stained by FM4–64) per cell was determined for the strains shown in A. For each experiment, 200 cells per strain were analyzed and grouped into the indicated categories. Two experiments were averaged. Bar, 5 μm.
Mentions: Absence of the vma phenotype makes vph1 mutants suitable for probing a role of V0 in vacuolar fusion in vivo. Mutation of fusion-relevant components frequently results in a fragmentation of vacuoles into multiple small vesicles in vivo (Raymond et al., 1992; Wada et al., 1992b). In some mutants, fragmentation does not occur, for example, after mutation of the v-SNARE Nyv1, Vtc1, or Vtc4 (Nichols et al., 1997; Muller et al., 2002). We assayed the in vivo effects of V-ATPase mutations by light microscopy after staining the vacuolar membranes with the red fluorescent vital dye FM4–64. Mutants lacking the vacuolar V0 subunit Vph1p showed numerous small vacuolar fragments which formed clusters (Fig. 1). This phenotype was observed with high frequency and resembles that of mutants in other genes with a function in the postdocking phase of vacuole fusion, such as Vtc3, Glc7 (protein phosphatase 1), calmodulin, or Vac8 (Peters and Mayer, 1998; Peters et al., 1999, 2001; Wang et al., 2001b; Muller et al., 2002). In contrast, mutation of Stv1, the Golgi/endosomal isoform of Vph1, did not result in vacuolar fragmentation (Fig. 1), nor did deletion of the V1 subunit Vma1p (unpublished data; with the strong caveat that this last mutant has the severe vma phenotype). Our results differ from those published in another study (Perzov et al., 2002) that reported vacuolar fragmentation for Δstv1 cells but not for Δvph1 cells. Due to this discrepancy, we generated Δvph1 mutants in three independent strain backgrounds and consistently observed vacuolar fragmentation. Currently, we have no explanation for this difference. However, our results are strongly corroborated by an unbiased microscopic screen for deletion mutants showing vacuolar fragmentation (Seeley et al., 2002). This genome wide screen identified mutants in four out of five Vo subunits as defective in vacuolar morphology, reporting a particularly strong phenotype for Δvph1 but none for Δstv1 or Δvma1.

Bottom Line: Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse.The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release.The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release.

View Article: PubMed Central - PubMed

Affiliation: Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tübingen, Germany.

ABSTRACT
Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.

Show MeSH
Related in: MedlinePlus