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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 activity of vacuoles from strains deficient in V1 sector (Δvma1) or in V0 sector (Δvph1). (A) Vacuoles from the indicated mutants (DKY 6281 background) were fused with wild-type vacuoles (BJ3505). Incubations were performed in the presence or absence of untreated cytosol (C) or of cytosol immunodepleted for Vma1p and Vma2p (dC). Fusion was assayed after 70 min at 27°C. The fusion activities of control reactions (asterisk) were set to 100%. The activity of the samples on ice was set to 0% (n = 5). Fusion activities of the wild-type–wild-type combination ranged from 2.2 to 4.1 U. (B) Western blot of equal protein amounts of cytosol (C) and immunodepleted cytosol (dC) against Vma1p, Vma2p, and the cytosolic marker protein phosphoglycerate kinase (PGK), verifying the absence of V1 sectors in the depleted cytosol.
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fig2: Fusion activity of vacuoles from strains deficient in V1 sector (Δvma1) or in V0 sector (Δvph1). (A) Vacuoles from the indicated mutants (DKY 6281 background) were fused with wild-type vacuoles (BJ3505). Incubations were performed in the presence or absence of untreated cytosol (C) or of cytosol immunodepleted for Vma1p and Vma2p (dC). Fusion was assayed after 70 min at 27°C. The fusion activities of control reactions (asterisk) were set to 100%. The activity of the samples on ice was set to 0% (n = 5). Fusion activities of the wild-type–wild-type combination ranged from 2.2 to 4.1 U. (B) Western blot of equal protein amounts of cytosol (C) and immunodepleted cytosol (dC) against Vma1p, Vma2p, and the cytosolic marker protein phosphoglycerate kinase (PGK), verifying the absence of V1 sectors in the depleted cytosol.

Mentions: In vivo experiments provide only indirect information about the proton uptake and fusion activities of the vacuoles. To overcome this limitation, we analyzed this aspect in an in vitro system reconstituting vacuolar fusion (Conradt et al., 1992). Vacuoles can be extracted from yeast spheroplasts after enzymatic digestion of the cell wall and gentle lysis by DEAE-dextrane, a treatment that leaves vacuoles intact and fusion competent. The isolated organelles fuse upon incubation with an ATP-regenerating system and cytosolic extracts. Since both V1 and V0 sectors are essential for proton translocation activity of the V-ATPase (Stevens and Forgac, 1997; Kane, 1999), we compared the fusion competence of vacuoles from V1 and V0 mutants, testing whether this V-ATPase activity was required for vacuole fusion. Vacuoles purified from the V0 mutant Δvph1 were deficient for in vitro fusion. The fusion defect was even observed if only one of the fusion partners was a Δvph1 vacuole and the other fusion partner was a wild-type vacuole (Fig. 2 A). This suggests that Vph1p is required for fusion on both fusion partners. Vacuoles from the V1 mutant Δvma1 fused with ∼50% of wild-type activity (Fig. 2 A). Addition of a cytosolic extract increased the fusion activity of Δvma1 vacuoles to 70%. This activation was not due to readdition of intact V1 subunits via the cytosolic extract because immunodepletion of V1 from these extracts (Fig. 2 B) did not diminish their activities (Fig. 2 A). Δvph1 vacuoles remained fusion incompetent under all conditions. Since both deletions of the V0 subunit Vph1 and deletion of the V1 subunit Vma1 completely abolish the pump activity of the V-ATPase (Stevens and Forgac, 1997; Kane, 1999), the difference in fusion activity between these strains is best explained by a function of Vph1p for vacuolar fusion that is independent of proton translocation.


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 activity of vacuoles from strains deficient in V1 sector (Δvma1) or in V0 sector (Δvph1). (A) Vacuoles from the indicated mutants (DKY 6281 background) were fused with wild-type vacuoles (BJ3505). Incubations were performed in the presence or absence of untreated cytosol (C) or of cytosol immunodepleted for Vma1p and Vma2p (dC). Fusion was assayed after 70 min at 27°C. The fusion activities of control reactions (asterisk) were set to 100%. The activity of the samples on ice was set to 0% (n = 5). Fusion activities of the wild-type–wild-type combination ranged from 2.2 to 4.1 U. (B) Western blot of equal protein amounts of cytosol (C) and immunodepleted cytosol (dC) against Vma1p, Vma2p, and the cytosolic marker protein phosphoglycerate kinase (PGK), verifying the absence of V1 sectors in the depleted cytosol.
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Related In: Results  -  Collection

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fig2: Fusion activity of vacuoles from strains deficient in V1 sector (Δvma1) or in V0 sector (Δvph1). (A) Vacuoles from the indicated mutants (DKY 6281 background) were fused with wild-type vacuoles (BJ3505). Incubations were performed in the presence or absence of untreated cytosol (C) or of cytosol immunodepleted for Vma1p and Vma2p (dC). Fusion was assayed after 70 min at 27°C. The fusion activities of control reactions (asterisk) were set to 100%. The activity of the samples on ice was set to 0% (n = 5). Fusion activities of the wild-type–wild-type combination ranged from 2.2 to 4.1 U. (B) Western blot of equal protein amounts of cytosol (C) and immunodepleted cytosol (dC) against Vma1p, Vma2p, and the cytosolic marker protein phosphoglycerate kinase (PGK), verifying the absence of V1 sectors in the depleted cytosol.
Mentions: In vivo experiments provide only indirect information about the proton uptake and fusion activities of the vacuoles. To overcome this limitation, we analyzed this aspect in an in vitro system reconstituting vacuolar fusion (Conradt et al., 1992). Vacuoles can be extracted from yeast spheroplasts after enzymatic digestion of the cell wall and gentle lysis by DEAE-dextrane, a treatment that leaves vacuoles intact and fusion competent. The isolated organelles fuse upon incubation with an ATP-regenerating system and cytosolic extracts. Since both V1 and V0 sectors are essential for proton translocation activity of the V-ATPase (Stevens and Forgac, 1997; Kane, 1999), we compared the fusion competence of vacuoles from V1 and V0 mutants, testing whether this V-ATPase activity was required for vacuole fusion. Vacuoles purified from the V0 mutant Δvph1 were deficient for in vitro fusion. The fusion defect was even observed if only one of the fusion partners was a Δvph1 vacuole and the other fusion partner was a wild-type vacuole (Fig. 2 A). This suggests that Vph1p is required for fusion on both fusion partners. Vacuoles from the V1 mutant Δvma1 fused with ∼50% of wild-type activity (Fig. 2 A). Addition of a cytosolic extract increased the fusion activity of Δvma1 vacuoles to 70%. This activation was not due to readdition of intact V1 subunits via the cytosolic extract because immunodepletion of V1 from these extracts (Fig. 2 B) did not diminish their activities (Fig. 2 A). Δvph1 vacuoles remained fusion incompetent under all conditions. Since both deletions of the V0 subunit Vph1 and deletion of the V1 subunit Vma1 completely abolish the pump activity of the V-ATPase (Stevens and Forgac, 1997; Kane, 1999), the difference in fusion activity between these strains is best explained by a function of Vph1p for vacuolar fusion that is independent of proton translocation.

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