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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 Ca2+ release during fusion. (A) Inhibition by antibodies to Vph1p. Standard fusion reactions were started, and Ca2+ was monitored continuously as described in the Materials and methods. Before the reactions were started, vacuoles had been preincubated with one of the indicated inhibitors, or with one of the following buffers only (3 min, 0°C): anti-Sec18p (2 μM), anti-Sec17p (2 μM), anti-Vph1p (20 μM), and nonimmune antibodies (20 μM). (B) Effect of vph1 deletion. Vacuoles were prepared from wild-type and Δvph1 cells and incubated under fusion conditions in the presence or absence of 5 μM Gdi1p. Ca2+ efflux was monitored as in A, and peak signals were plotted.
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fig9: Vacuolar Ca2+ release during fusion. (A) Inhibition by antibodies to Vph1p. Standard fusion reactions were started, and Ca2+ was monitored continuously as described in the Materials and methods. Before the reactions were started, vacuoles had been preincubated with one of the indicated inhibitors, or with one of the following buffers only (3 min, 0°C): anti-Sec18p (2 μM), anti-Sec17p (2 μM), anti-Vph1p (20 μM), and nonimmune antibodies (20 μM). (B) Effect of vph1 deletion. Vacuoles were prepared from wild-type and Δvph1 cells and incubated under fusion conditions in the presence or absence of 5 μM Gdi1p. Ca2+ efflux was monitored as in A, and peak signals were plotted.

Mentions: The relationship of Ca2+ release and Vph1p function in fusion was further characterized by monitoring Ca2+ release in the course of fusion. Fusion reactions were run in the presence of aequorin and coelenterazine (Muller et al., 2001). Aequorin is a protein that oxidizes its substrate coelenterazine in a Ca2+-dependent fashion, emitting light (Shimomura et al., 1993). This permits continuous recording of Ca2+ levels in the buffer of ongoing fusion reactions. In agreement with previous results obtained with a different approach (Peters and Mayer, 1998), treatments preventing priming, such as antibodies to Sec18p/NSF or Sec17p/α-SNAP, suppressed the release of Ca2+ from the vacuolar lumen (Fig. 9 A). Docking inhibitors, such as antibodies to the t-SNAREs Vam3p and Vam7p had the same effect (not depicted). Surprisingly, antibodies to Vph1p also inhibited Ca2+ release, suggesting that either Vph1p or V0 itself might be involved in Ca2+ release or that Vph1p might be coupled to the Ca2+ channel. In both cases, adding an antibody to Vph1p might block a conformational change required for activation of the channel. Only in the first case, however, should deletion of Vph1p, which eliminates the vacuolar V0 sector as a whole (Stevens and Forgac, 1997; Kane and Parra, 2000), abolish Ca2+ release. This was not the case. Δvph1 vacuoles showed Ca2+efflux just as their wild-type counterparts (Fig. 9 B). Efflux was sensitive to the Rab-GTPase inhibitor Gdi1p, demonstrating that it depended on docking and was triggered by the authentic fusion pathway. This permits several conclusions. First, it suggests that the fusion defect of Δvph1 vacuoles is not due to the inability to release calcium. Second, Vph1p itself is not part of the Ca2+-releasing channel. Third, Vph1p may be coupled to the Ca2+ channel, leaving calcium release intact if Vph1p is absent but blocking it if Vph1p is inactivated by an antibody.


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 Ca2+ release during fusion. (A) Inhibition by antibodies to Vph1p. Standard fusion reactions were started, and Ca2+ was monitored continuously as described in the Materials and methods. Before the reactions were started, vacuoles had been preincubated with one of the indicated inhibitors, or with one of the following buffers only (3 min, 0°C): anti-Sec18p (2 μM), anti-Sec17p (2 μM), anti-Vph1p (20 μM), and nonimmune antibodies (20 μM). (B) Effect of vph1 deletion. Vacuoles were prepared from wild-type and Δvph1 cells and incubated under fusion conditions in the presence or absence of 5 μM Gdi1p. Ca2+ efflux was monitored as in A, and peak signals were plotted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172786&req=5

fig9: Vacuolar Ca2+ release during fusion. (A) Inhibition by antibodies to Vph1p. Standard fusion reactions were started, and Ca2+ was monitored continuously as described in the Materials and methods. Before the reactions were started, vacuoles had been preincubated with one of the indicated inhibitors, or with one of the following buffers only (3 min, 0°C): anti-Sec18p (2 μM), anti-Sec17p (2 μM), anti-Vph1p (20 μM), and nonimmune antibodies (20 μM). (B) Effect of vph1 deletion. Vacuoles were prepared from wild-type and Δvph1 cells and incubated under fusion conditions in the presence or absence of 5 μM Gdi1p. Ca2+ efflux was monitored as in A, and peak signals were plotted.
Mentions: The relationship of Ca2+ release and Vph1p function in fusion was further characterized by monitoring Ca2+ release in the course of fusion. Fusion reactions were run in the presence of aequorin and coelenterazine (Muller et al., 2001). Aequorin is a protein that oxidizes its substrate coelenterazine in a Ca2+-dependent fashion, emitting light (Shimomura et al., 1993). This permits continuous recording of Ca2+ levels in the buffer of ongoing fusion reactions. In agreement with previous results obtained with a different approach (Peters and Mayer, 1998), treatments preventing priming, such as antibodies to Sec18p/NSF or Sec17p/α-SNAP, suppressed the release of Ca2+ from the vacuolar lumen (Fig. 9 A). Docking inhibitors, such as antibodies to the t-SNAREs Vam3p and Vam7p had the same effect (not depicted). Surprisingly, antibodies to Vph1p also inhibited Ca2+ release, suggesting that either Vph1p or V0 itself might be involved in Ca2+ release or that Vph1p might be coupled to the Ca2+ channel. In both cases, adding an antibody to Vph1p might block a conformational change required for activation of the channel. Only in the first case, however, should deletion of Vph1p, which eliminates the vacuolar V0 sector as a whole (Stevens and Forgac, 1997; Kane and Parra, 2000), abolish Ca2+ release. This was not the case. Δvph1 vacuoles showed Ca2+efflux just as their wild-type counterparts (Fig. 9 B). Efflux was sensitive to the Rab-GTPase inhibitor Gdi1p, demonstrating that it depended on docking and was triggered by the authentic fusion pathway. This permits several conclusions. First, it suggests that the fusion defect of Δvph1 vacuoles is not due to the inability to release calcium. Second, Vph1p itself is not part of the Ca2+-releasing channel. Third, Vph1p may be coupled to the Ca2+ channel, leaving calcium release intact if Vph1p is absent but blocking it if Vph1p is inactivated by an antibody.

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