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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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Fusion of vacuoles without V1 sector is inhibited by anti-Vph1p. (A) Vacuoles were extracted with SCN−/ATP to remove V1. Fusion activity was assayed in the absence or presence of the indicated inhibitors as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 0.75 to 1.4 U. (B) Apparent proton pumping activity of untreated or stripped vacuoles was determined as described in Materials and methods. The activity of untreated vacuoles was set to 100%. Inhibitors were GTPγS (2 mM), anti-Vph1p (20 μM), nonimmune antibodies (20 μM), and anti-Sec18p (2 μM).
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fig7: Fusion of vacuoles without V1 sector is inhibited by anti-Vph1p. (A) Vacuoles were extracted with SCN−/ATP to remove V1. Fusion activity was assayed in the absence or presence of the indicated inhibitors as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 0.75 to 1.4 U. (B) Apparent proton pumping activity of untreated or stripped vacuoles was determined as described in Materials and methods. The activity of untreated vacuoles was set to 100%. Inhibitors were GTPγS (2 mM), anti-Vph1p (20 μM), nonimmune antibodies (20 μM), and anti-Sec18p (2 μM).

Mentions: As described for the Δvph1 mutants above, we tested whether antibodies to Vph1p would interfere with the apparent vacuolar proton uptake. This was not the case, although the antibodies blocked fusion (Fig. 6). The lack of effect of anti-Vph1p on proton uptake is probably due to the fact that the antibodies recognize only solitary V0 sectors but not the assembled V0/V1 holoenzyme (unpublished data). The antibodies were also tested on vacuoles from which the V1 sector had been removed by cold extraction with KSCN and ATP, a treatment that leaves the V0 sector in the membranes (Arai et al., 1989; Moriyama and Nelson, 1989; Adachi et al., 1990; Peters et al., 2001) but inactivates V-ATPase–dependent proton translocation (Fig. 7 B). Fusion of such vacuoles was inhibited by anti-Vph1p as efficiently as by GTPγS or antibodies to Sec18p/NSF (Fig. 7 A), two established antagonists of vacuole fusion (Haas et al., 1994; Mayer et al., 1996). Thus, antibodies to the cytosolic part of Vph1p provide an additional tool to separate the fusion function of V0 from its role in proton pumping.


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)

Fusion of vacuoles without V1 sector is inhibited by anti-Vph1p. (A) Vacuoles were extracted with SCN−/ATP to remove V1. Fusion activity was assayed in the absence or presence of the indicated inhibitors as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 0.75 to 1.4 U. (B) Apparent proton pumping activity of untreated or stripped vacuoles was determined as described in Materials and methods. The activity of untreated vacuoles was set to 100%. Inhibitors were GTPγS (2 mM), anti-Vph1p (20 μM), nonimmune antibodies (20 μM), and anti-Sec18p (2 μM).
© Copyright Policy
Related In: Results  -  Collection

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fig7: Fusion of vacuoles without V1 sector is inhibited by anti-Vph1p. (A) Vacuoles were extracted with SCN−/ATP to remove V1. Fusion activity was assayed in the absence or presence of the indicated inhibitors as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 0.75 to 1.4 U. (B) Apparent proton pumping activity of untreated or stripped vacuoles was determined as described in Materials and methods. The activity of untreated vacuoles was set to 100%. Inhibitors were GTPγS (2 mM), anti-Vph1p (20 μM), nonimmune antibodies (20 μM), and anti-Sec18p (2 μM).
Mentions: As described for the Δvph1 mutants above, we tested whether antibodies to Vph1p would interfere with the apparent vacuolar proton uptake. This was not the case, although the antibodies blocked fusion (Fig. 6). The lack of effect of anti-Vph1p on proton uptake is probably due to the fact that the antibodies recognize only solitary V0 sectors but not the assembled V0/V1 holoenzyme (unpublished data). The antibodies were also tested on vacuoles from which the V1 sector had been removed by cold extraction with KSCN and ATP, a treatment that leaves the V0 sector in the membranes (Arai et al., 1989; Moriyama and Nelson, 1989; Adachi et al., 1990; Peters et al., 2001) but inactivates V-ATPase–dependent proton translocation (Fig. 7 B). Fusion of such vacuoles was inhibited by anti-Vph1p as efficiently as by GTPγS or antibodies to Sec18p/NSF (Fig. 7 A), two established antagonists of vacuole fusion (Haas et al., 1994; Mayer et al., 1996). Thus, antibodies to the cytosolic part of Vph1p provide an additional tool to separate the fusion function of V0 from its role in proton pumping.

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