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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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Detection of V-ATPase–independent proton uptake activity by acridine orange. (A) Concentration dependence of the assay. Proton uptake activity of the vacuoles was assayed by measuring acridine orange absorption. Fusion reactions were started in the presence of 15 μM acridine orange and different vacuole concentrations to vary the dye to vacuole ratio. Where indicated (start), the ATP regenerating system and concanamycin A had been added. At the end of the assay period, FCCP (30 μM) was added to dissipate the proton gradient. (B) Assay of mutant vacuoles. Vacuoles from wild-type, Δvph1, and Δvma2 cells were assayed for proton uptake activity as in A. 54 pmol acridine orange (final concentration 15 μM) were used per μg vacuoles.
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fig3: Detection of V-ATPase–independent proton uptake activity by acridine orange. (A) Concentration dependence of the assay. Proton uptake activity of the vacuoles was assayed by measuring acridine orange absorption. Fusion reactions were started in the presence of 15 μM acridine orange and different vacuole concentrations to vary the dye to vacuole ratio. Where indicated (start), the ATP regenerating system and concanamycin A had been added. At the end of the assay period, FCCP (30 μM) was added to dissipate the proton gradient. (B) Assay of mutant vacuoles. Vacuoles from wild-type, Δvph1, and Δvma2 cells were assayed for proton uptake activity as in A. 54 pmol acridine orange (final concentration 15 μM) were used per μg vacuoles.

Mentions: Since vacuole fusion depends on a pmf across the vacuolar membrane (Conradt et al., 1994; Ungermann et al., 1999b) and the V-ATPase is the major vacuolar proton pump (Stevens and Forgac, 1997), we asked whether the fusion activity of Δvph1 vacuoles could be limited by lack of a pmf. We tested whether Δvph1 vacuoles are able to generate a basal pmf by a V-ATPase–independent pathway and whether this pmf would be above the threshold required for fusion. V-ATPase–independent antiport mechanisms can reconstitute a basal proton uptake activity by antiport of, for example, amino acids and protons (Ohsumi and Anraku, 1981, 1983; Sato et al., 1984; Klionsky et al., 1990; Wada et al., 1992a; Wada and Anraku, 1994) and/or by combination of an active vacuolar Ca2+ pump and a Ca2+/H+ antiporter (Ohsumi and Anraku, 1983; Cunningham and Fink, 1994; Forster and Kane, 2000). The pmf as such is important to drive fusion, but for the purpose of our experiments it is irrelevant whether proton uptake occurs via the V-ATPase or alternative mechanisms. We measured the apparent proton uptake activity of whole vacuoles under the conditions of our fusion reaction by following the change in absorbance of acridine orange at 491 and 540 nm, a common assay for the vacuolar pmf (Gluck et al., 1982). Since acridine orange is also fluorescent, we could monitor its distribution in the membrane preparation by fluorescence microscopy. At least 92% of the dye localized to structures that, by size and morphology, could be identified as vacuoles (unpublished data). Therefore, acridine orange will mostly report changes of the pmf on vacuoles. At standard dye to membrane ratios wild-type vacuoles showed a clear ATP-dependent decrease in the absorbance signal, indicating that a pmf had formed (Fig. 3 A, bottom panel). Addition of the protonophor carbonylcyanide-4-trifluormethoxyphenylhydrazon (FCCP) at the end of the assay period collapsed the pmf and restored the signal to the initial values. No signal was observed in the absence of ATP or after addition of even low concentrations (0.1 μM) of the V-ATPase inhibitor concanamycin A. Concanamycin A blocks V-ATPase–dependent H+ translocation at nanomolar concentrations (Drose and Altendorf, 1997). Our observation is consistent with earlier results from various other groups using subvacuolar vesicles (Drose and Altendorf, 1997). These studies demonstrated that the V-ATPase was the main source of the vacuolar pmf and could be completely inhibited by bafilomycin A or concanamycin A. At lower dye to vacuole ratios (Fig. 3 A, top two panels), however, the assay was more sensitive and could still detect a pmf in the presence of 0.1 μM concanamycin A. 1 and 5 μM of the inhibitor reduced the signal further and yielded overlapping curves, suggesting that these concentrations had saturated the system and that V-ATPase had been completely inhibited. Nevertheless, significant ATP-dependent proton uptake activity remained even at these high concentrations. This concanamycin-insensitive activity was comparable to the levels of ATP-dependent proton uptake observable with vacuoles prepared from V1 (Δvma2) or V0 (Δvph1) mutant cells (Fig. 3 B). The signal in V-ATPase mutant vacuoles could not be further reduced by concanamycin A. Therefore, this signal appears to reflect the V-ATPase–independent part of vacuolar proton uptake.


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)

Detection of V-ATPase–independent proton uptake activity by acridine orange. (A) Concentration dependence of the assay. Proton uptake activity of the vacuoles was assayed by measuring acridine orange absorption. Fusion reactions were started in the presence of 15 μM acridine orange and different vacuole concentrations to vary the dye to vacuole ratio. Where indicated (start), the ATP regenerating system and concanamycin A had been added. At the end of the assay period, FCCP (30 μM) was added to dissipate the proton gradient. (B) Assay of mutant vacuoles. Vacuoles from wild-type, Δvph1, and Δvma2 cells were assayed for proton uptake activity as in A. 54 pmol acridine orange (final concentration 15 μM) were used per μg vacuoles.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Detection of V-ATPase–independent proton uptake activity by acridine orange. (A) Concentration dependence of the assay. Proton uptake activity of the vacuoles was assayed by measuring acridine orange absorption. Fusion reactions were started in the presence of 15 μM acridine orange and different vacuole concentrations to vary the dye to vacuole ratio. Where indicated (start), the ATP regenerating system and concanamycin A had been added. At the end of the assay period, FCCP (30 μM) was added to dissipate the proton gradient. (B) Assay of mutant vacuoles. Vacuoles from wild-type, Δvph1, and Δvma2 cells were assayed for proton uptake activity as in A. 54 pmol acridine orange (final concentration 15 μM) were used per μg vacuoles.
Mentions: Since vacuole fusion depends on a pmf across the vacuolar membrane (Conradt et al., 1994; Ungermann et al., 1999b) and the V-ATPase is the major vacuolar proton pump (Stevens and Forgac, 1997), we asked whether the fusion activity of Δvph1 vacuoles could be limited by lack of a pmf. We tested whether Δvph1 vacuoles are able to generate a basal pmf by a V-ATPase–independent pathway and whether this pmf would be above the threshold required for fusion. V-ATPase–independent antiport mechanisms can reconstitute a basal proton uptake activity by antiport of, for example, amino acids and protons (Ohsumi and Anraku, 1981, 1983; Sato et al., 1984; Klionsky et al., 1990; Wada et al., 1992a; Wada and Anraku, 1994) and/or by combination of an active vacuolar Ca2+ pump and a Ca2+/H+ antiporter (Ohsumi and Anraku, 1983; Cunningham and Fink, 1994; Forster and Kane, 2000). The pmf as such is important to drive fusion, but for the purpose of our experiments it is irrelevant whether proton uptake occurs via the V-ATPase or alternative mechanisms. We measured the apparent proton uptake activity of whole vacuoles under the conditions of our fusion reaction by following the change in absorbance of acridine orange at 491 and 540 nm, a common assay for the vacuolar pmf (Gluck et al., 1982). Since acridine orange is also fluorescent, we could monitor its distribution in the membrane preparation by fluorescence microscopy. At least 92% of the dye localized to structures that, by size and morphology, could be identified as vacuoles (unpublished data). Therefore, acridine orange will mostly report changes of the pmf on vacuoles. At standard dye to membrane ratios wild-type vacuoles showed a clear ATP-dependent decrease in the absorbance signal, indicating that a pmf had formed (Fig. 3 A, bottom panel). Addition of the protonophor carbonylcyanide-4-trifluormethoxyphenylhydrazon (FCCP) at the end of the assay period collapsed the pmf and restored the signal to the initial values. No signal was observed in the absence of ATP or after addition of even low concentrations (0.1 μM) of the V-ATPase inhibitor concanamycin A. Concanamycin A blocks V-ATPase–dependent H+ translocation at nanomolar concentrations (Drose and Altendorf, 1997). Our observation is consistent with earlier results from various other groups using subvacuolar vesicles (Drose and Altendorf, 1997). These studies demonstrated that the V-ATPase was the main source of the vacuolar pmf and could be completely inhibited by bafilomycin A or concanamycin A. At lower dye to vacuole ratios (Fig. 3 A, top two panels), however, the assay was more sensitive and could still detect a pmf in the presence of 0.1 μM concanamycin A. 1 and 5 μM of the inhibitor reduced the signal further and yielded overlapping curves, suggesting that these concentrations had saturated the system and that V-ATPase had been completely inhibited. Nevertheless, significant ATP-dependent proton uptake activity remained even at these high concentrations. This concanamycin-insensitive activity was comparable to the levels of ATP-dependent proton uptake observable with vacuoles prepared from V1 (Δvma2) or V0 (Δvph1) mutant cells (Fig. 3 B). The signal in V-ATPase mutant vacuoles could not be further reduced by concanamycin A. Therefore, this signal appears to reflect the V-ATPase–independent part of vacuolar proton uptake.

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