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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 and apparent proton translocation activity of vacuoles from vph1 deletion strains (Δvph1). (A) Fusion and apparent proton translocation activity (B) were examined in parallel fusion reactions using wild-type or Δvph1 vacuoles in the presence or absence of concanamycin A (Conc.A; 1 μM) or FCCP (30 μM). Fusion and pump activities of wild-type vacuoles (asterisk) were set to 100% (n = 3). Fusion activities of these control samples ranged from 2.5 to 3.8 U.
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fig4: Fusion and apparent proton translocation activity of vacuoles from vph1 deletion strains (Δvph1). (A) Fusion and apparent proton translocation activity (B) were examined in parallel fusion reactions using wild-type or Δvph1 vacuoles in the presence or absence of concanamycin A (Conc.A; 1 μM) or FCCP (30 μM). Fusion and pump activities of wild-type vacuoles (asterisk) were set to 100% (n = 3). Fusion activities of these control samples ranged from 2.5 to 3.8 U.

Mentions: Next, we asked whether the V-ATPase–independent proton uptake activity could suffice to drive fusion. We compared the fusion and proton translocation activities of Δvph1 vacuoles to those of wild-type vacuoles in which V-ATPase pump activity had been pharmacologically suppressed with concanamycin A. In this case, we measured apparent proton uptake activity by following alkalinization of the medium with the pH-sensitive dye 2′7′-bis-[2-carboxyethyl]-5-[and 6]-carboxyfluorescein (BCECF; Peters et al., 2001). Unlike acridine orange, which inserts into the vacuolar membrane (unpublished data), this dye is coupled to high molecular weight dextrane, does not interfere with vacuole fusion, and permits assay of fusion and proton translocation in the same samples. In this assay, the apparent proton translocation activity of Δvph1 vacuoles was equal to that of wild-type vacuoles treated with 1 μM concanamycin A (Fig. 4 B). As in the acridine orange assay, this signal was ATP dependent and FCCP sensitive, suggesting that it reflected the same V-ATPase–independent component of vacuolar proton uptake. Comparison of the fusion activities revealed that Δvph1 vacuoles did not fuse, but wild-type vacuoles with 1 μM concanamycin A retained full fusion activity (Fig. 4 A). FCCP dissipates the proton gradient across the vacuolar membrane, eliminating both its V-ATPase–dependent and V-ATPase–independent components. In agreement with earlier studies (Conradt et al., 1994; Mayer et al., 1996; Ungermann et al., 1999b), FCCP inhibited fusion. Together, vacuole fusion seems to depend on a pmf across the vacuolar membrane but not on V-ATPase proton pump activity. Hence, the fusion defect of Δvph1 vacuoles cannot be explained by a reduction in the apparent proton uptake activity, supporting a direct function of V0 in vacuole fusion.


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 and apparent proton translocation activity of vacuoles from vph1 deletion strains (Δvph1). (A) Fusion and apparent proton translocation activity (B) were examined in parallel fusion reactions using wild-type or Δvph1 vacuoles in the presence or absence of concanamycin A (Conc.A; 1 μM) or FCCP (30 μM). Fusion and pump activities of wild-type vacuoles (asterisk) were set to 100% (n = 3). Fusion activities of these control samples ranged from 2.5 to 3.8 U.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Fusion and apparent proton translocation activity of vacuoles from vph1 deletion strains (Δvph1). (A) Fusion and apparent proton translocation activity (B) were examined in parallel fusion reactions using wild-type or Δvph1 vacuoles in the presence or absence of concanamycin A (Conc.A; 1 μM) or FCCP (30 μM). Fusion and pump activities of wild-type vacuoles (asterisk) were set to 100% (n = 3). Fusion activities of these control samples ranged from 2.5 to 3.8 U.
Mentions: Next, we asked whether the V-ATPase–independent proton uptake activity could suffice to drive fusion. We compared the fusion and proton translocation activities of Δvph1 vacuoles to those of wild-type vacuoles in which V-ATPase pump activity had been pharmacologically suppressed with concanamycin A. In this case, we measured apparent proton uptake activity by following alkalinization of the medium with the pH-sensitive dye 2′7′-bis-[2-carboxyethyl]-5-[and 6]-carboxyfluorescein (BCECF; Peters et al., 2001). Unlike acridine orange, which inserts into the vacuolar membrane (unpublished data), this dye is coupled to high molecular weight dextrane, does not interfere with vacuole fusion, and permits assay of fusion and proton translocation in the same samples. In this assay, the apparent proton translocation activity of Δvph1 vacuoles was equal to that of wild-type vacuoles treated with 1 μM concanamycin A (Fig. 4 B). As in the acridine orange assay, this signal was ATP dependent and FCCP sensitive, suggesting that it reflected the same V-ATPase–independent component of vacuolar proton uptake. Comparison of the fusion activities revealed that Δvph1 vacuoles did not fuse, but wild-type vacuoles with 1 μM concanamycin A retained full fusion activity (Fig. 4 A). FCCP dissipates the proton gradient across the vacuolar membrane, eliminating both its V-ATPase–dependent and V-ATPase–independent components. In agreement with earlier studies (Conradt et al., 1994; Mayer et al., 1996; Ungermann et al., 1999b), FCCP inhibited fusion. Together, vacuole fusion seems to depend on a pmf across the vacuolar membrane but not on V-ATPase proton pump activity. Hence, the fusion defect of Δvph1 vacuoles cannot be explained by a reduction in the apparent proton uptake activity, supporting a direct function of V0 in vacuole fusion.

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