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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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Kinetic resolution of Vph1p requirement for fusion. (A) Standard fusion reactions without cytosol were started at 27°C. At the indicated times, inhibitors or control buffer were added. The samples were left on ice for 10 min. Then, they were transferred to 27°C or left on ice for the remainder of the 70-min reaction period. After 70 min, fusion activity was assayed. (B) Trans-SNARE pairing. Fusion reactions with vacuoles from strains SBY521 (Δvam3 Δvph1) or no. 418 (Δvam3 VPH1) and no. 120 (Δnyv1 VPH1) were incubated (50 min, 27°C) with the indicated inhibitors at 27°C or left on ice. Then, the membranes were reisolated, solubilized, and assayed for trans-SNARE complexes by determining the amounts of the v-SNARE Nyv1p coimmunoprecipitating with the t-SNARE Vam3p. (C) Standard fusion reactions without cytosol were incubated at 27°C in the presence of 1 mM MgCl2 and 5 mM BAPTA for 30 min. The reactions were chilled on ice, and vacuoles were reisolated (10,000 g, 2 min, 2°C). Vacuoles were resuspended in fusion buffer with cytosol and 200 μM CaCl2 but without ATP. Aliquots were preincubated for 5 min on ice with the indicated inhibitors and incubated further for 70 min at 27°C. Fusion activities were assayed and plotted as in the legend to Fig. 2 (n = 3). Activities of the control sample ranged from 1.1 to 1.9 U. Inhibitors were BAPTA (5 mM), GTPγS (2 mM), anti-Vam3p (2 μM), Gdi1p (5 μM), anti-Vph1p (20 μM), and FCCP (30 μM).
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fig8: Kinetic resolution of Vph1p requirement for fusion. (A) Standard fusion reactions without cytosol were started at 27°C. At the indicated times, inhibitors or control buffer were added. The samples were left on ice for 10 min. Then, they were transferred to 27°C or left on ice for the remainder of the 70-min reaction period. After 70 min, fusion activity was assayed. (B) Trans-SNARE pairing. Fusion reactions with vacuoles from strains SBY521 (Δvam3 Δvph1) or no. 418 (Δvam3 VPH1) and no. 120 (Δnyv1 VPH1) were incubated (50 min, 27°C) with the indicated inhibitors at 27°C or left on ice. Then, the membranes were reisolated, solubilized, and assayed for trans-SNARE complexes by determining the amounts of the v-SNARE Nyv1p coimmunoprecipitating with the t-SNARE Vam3p. (C) Standard fusion reactions without cytosol were incubated at 27°C in the presence of 1 mM MgCl2 and 5 mM BAPTA for 30 min. The reactions were chilled on ice, and vacuoles were reisolated (10,000 g, 2 min, 2°C). Vacuoles were resuspended in fusion buffer with cytosol and 200 μM CaCl2 but without ATP. Aliquots were preincubated for 5 min on ice with the indicated inhibitors and incubated further for 70 min at 27°C. Fusion activities were assayed and plotted as in the legend to Fig. 2 (n = 3). Activities of the control sample ranged from 1.1 to 1.9 U. Inhibitors were BAPTA (5 mM), GTPγS (2 mM), anti-Vam3p (2 μM), Gdi1p (5 μM), anti-Vph1p (20 μM), and FCCP (30 μM).

Mentions: The antibodies to Vph1p were used for kinetic analyses in order to explore which stage of vacuole fusion required Vph1p. First, we tested when the reaction became resistant to anti-Vph1p (Fig. 8 A). The majority of vacuoles completes distinct reaction steps within defined intervals and becomes resistant to inhibitors of these steps (Conradt et al., 1994; Mayer et al., 1996), revealing the sequence of events. Inhibitors were added to an aliquot of an ongoing fusion reaction at different times after the start point. Then, the incubation was continued until the end of a standard fusion period (70 min). Another aliquot was chilled to stop fusion at the time of inhibitor addition. All inhibitors abolished fusion when added at the start of the reaction (Fig. 8 A). After 35 min, the reaction was resistant to antibodies to the t-SNARE Vam3p, indicating the completion of docking (Mayer and Wickner, 1997; Ungermann et al., 1998b). The inhibition curve for anti-Vph1p lagged significantly behind that for anti-Vam3p, suggesting that Vph1p was required past the docking stage. This behavior resembled the inhibition curves obtained previously with calmodulin antagonists or with the Ca2+ chelator BAPTA, which inhibit completion of fusion after the docking step (Peters and Mayer, 1998; Ungermann et al., 1999b).


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)

Kinetic resolution of Vph1p requirement for fusion. (A) Standard fusion reactions without cytosol were started at 27°C. At the indicated times, inhibitors or control buffer were added. The samples were left on ice for 10 min. Then, they were transferred to 27°C or left on ice for the remainder of the 70-min reaction period. After 70 min, fusion activity was assayed. (B) Trans-SNARE pairing. Fusion reactions with vacuoles from strains SBY521 (Δvam3 Δvph1) or no. 418 (Δvam3 VPH1) and no. 120 (Δnyv1 VPH1) were incubated (50 min, 27°C) with the indicated inhibitors at 27°C or left on ice. Then, the membranes were reisolated, solubilized, and assayed for trans-SNARE complexes by determining the amounts of the v-SNARE Nyv1p coimmunoprecipitating with the t-SNARE Vam3p. (C) Standard fusion reactions without cytosol were incubated at 27°C in the presence of 1 mM MgCl2 and 5 mM BAPTA for 30 min. The reactions were chilled on ice, and vacuoles were reisolated (10,000 g, 2 min, 2°C). Vacuoles were resuspended in fusion buffer with cytosol and 200 μM CaCl2 but without ATP. Aliquots were preincubated for 5 min on ice with the indicated inhibitors and incubated further for 70 min at 27°C. Fusion activities were assayed and plotted as in the legend to Fig. 2 (n = 3). Activities of the control sample ranged from 1.1 to 1.9 U. Inhibitors were BAPTA (5 mM), GTPγS (2 mM), anti-Vam3p (2 μM), Gdi1p (5 μM), anti-Vph1p (20 μM), and FCCP (30 μM).
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fig8: Kinetic resolution of Vph1p requirement for fusion. (A) Standard fusion reactions without cytosol were started at 27°C. At the indicated times, inhibitors or control buffer were added. The samples were left on ice for 10 min. Then, they were transferred to 27°C or left on ice for the remainder of the 70-min reaction period. After 70 min, fusion activity was assayed. (B) Trans-SNARE pairing. Fusion reactions with vacuoles from strains SBY521 (Δvam3 Δvph1) or no. 418 (Δvam3 VPH1) and no. 120 (Δnyv1 VPH1) were incubated (50 min, 27°C) with the indicated inhibitors at 27°C or left on ice. Then, the membranes were reisolated, solubilized, and assayed for trans-SNARE complexes by determining the amounts of the v-SNARE Nyv1p coimmunoprecipitating with the t-SNARE Vam3p. (C) Standard fusion reactions without cytosol were incubated at 27°C in the presence of 1 mM MgCl2 and 5 mM BAPTA for 30 min. The reactions were chilled on ice, and vacuoles were reisolated (10,000 g, 2 min, 2°C). Vacuoles were resuspended in fusion buffer with cytosol and 200 μM CaCl2 but without ATP. Aliquots were preincubated for 5 min on ice with the indicated inhibitors and incubated further for 70 min at 27°C. Fusion activities were assayed and plotted as in the legend to Fig. 2 (n = 3). Activities of the control sample ranged from 1.1 to 1.9 U. Inhibitors were BAPTA (5 mM), GTPγS (2 mM), anti-Vam3p (2 μM), Gdi1p (5 μM), anti-Vph1p (20 μM), and FCCP (30 μM).
Mentions: The antibodies to Vph1p were used for kinetic analyses in order to explore which stage of vacuole fusion required Vph1p. First, we tested when the reaction became resistant to anti-Vph1p (Fig. 8 A). The majority of vacuoles completes distinct reaction steps within defined intervals and becomes resistant to inhibitors of these steps (Conradt et al., 1994; Mayer et al., 1996), revealing the sequence of events. Inhibitors were added to an aliquot of an ongoing fusion reaction at different times after the start point. Then, the incubation was continued until the end of a standard fusion period (70 min). Another aliquot was chilled to stop fusion at the time of inhibitor addition. All inhibitors abolished fusion when added at the start of the reaction (Fig. 8 A). After 35 min, the reaction was resistant to antibodies to the t-SNARE Vam3p, indicating the completion of docking (Mayer and Wickner, 1997; Ungermann et al., 1998b). The inhibition curve for anti-Vph1p lagged significantly behind that for anti-Vam3p, suggesting that Vph1p was required past the docking stage. This behavior resembled the inhibition curves obtained previously with calmodulin antagonists or with the Ca2+ chelator BAPTA, which inhibit completion of fusion after the docking step (Peters and Mayer, 1998; Ungermann et al., 1999b).

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