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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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Inhibition by antibodies to Vph1p. (A) Different concentrations of affinity-purified antibodies to Vph1p (from goat) were added to a standard fusion reaction without cytosol. The reaction was started after preincubation for 10 min on ice by adding ATP. Fusion was assayed after 70 min at 27°C. (B) Nonimmune antibodies do not interfere with vacuole fusion. Standard fusion reactions were run with 20 μM IgG purified from nonimmune or anti-Vph1p sera from goats, or with control buffer only, and assayed after 70 min at 27°C (n = 6). (C) Inhibition of fusion by Fab fragments of antibodies to Vph1p. Standard fusion reactions were incubated with Fab fragments derived either from nonimmune antibodies or from antibodies to Vph1p. Fusion was assayed as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 3.2 to 5.1 U, and those of ice values ranged from 0.2 to 0.3 U. Inhibitor concentrations were 45 μM anti-Vph1p Fab and 45 μM nonimmune Fab.
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fig5: Inhibition by antibodies to Vph1p. (A) Different concentrations of affinity-purified antibodies to Vph1p (from goat) were added to a standard fusion reaction without cytosol. The reaction was started after preincubation for 10 min on ice by adding ATP. Fusion was assayed after 70 min at 27°C. (B) Nonimmune antibodies do not interfere with vacuole fusion. Standard fusion reactions were run with 20 μM IgG purified from nonimmune or anti-Vph1p sera from goats, or with control buffer only, and assayed after 70 min at 27°C (n = 6). (C) Inhibition of fusion by Fab fragments of antibodies to Vph1p. Standard fusion reactions were incubated with Fab fragments derived either from nonimmune antibodies or from antibodies to Vph1p. Fusion was assayed as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 3.2 to 5.1 U, and those of ice values ranged from 0.2 to 0.3 U. Inhibitor concentrations were 45 μM anti-Vph1p Fab and 45 μM nonimmune Fab.

Mentions: We sought to specifically interfere with the fusion relevant aspect of V0. Available low molecular weight V-ATPase inhibitors affect only the proton translocation function of the V-ATPase, (e.g., concanamycin A) and/or are slowly reacting and of unsatisfactory specificity (e.g., dicyclohexylcarbodiimide). We raised antibodies to V0 subunits in order to generate highly specific agents that might interfere with V0 function and be suitable for analysis of the fusion reaction. We immunized goats with the recombinantly expressed and purified NH2-terminal cytosolic part of Vph1p and prepared affinity-purified polyclonal antibodies from the sera. If titrated into in vitro fusion reactions, these antibodies inhibited fusion in a concentration-dependent manner (IC50= 6 μM; Fig. 5 A). Nonimmune antibodies had no effect at comparable concentrations (Fig. 5 B). To exclude potential interference due to the divalent nature of whole IgG, we also prepared monovalent Fab fragments from these antibodies. They also inhibited fusion at concentrations comparable to those observed for whole IgGs (Fig. 5 C), matching our general experience for many other fusion relevant components: the inhibitory potency of whole IgGs and Fab fragments is not significantly different (Haas et al., 1995; Haas and Wickner, 1996; Mayer and Wickner, 1997; Nichols et al., 1997).


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)

Inhibition by antibodies to Vph1p. (A) Different concentrations of affinity-purified antibodies to Vph1p (from goat) were added to a standard fusion reaction without cytosol. The reaction was started after preincubation for 10 min on ice by adding ATP. Fusion was assayed after 70 min at 27°C. (B) Nonimmune antibodies do not interfere with vacuole fusion. Standard fusion reactions were run with 20 μM IgG purified from nonimmune or anti-Vph1p sera from goats, or with control buffer only, and assayed after 70 min at 27°C (n = 6). (C) Inhibition of fusion by Fab fragments of antibodies to Vph1p. Standard fusion reactions were incubated with Fab fragments derived either from nonimmune antibodies or from antibodies to Vph1p. Fusion was assayed as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 3.2 to 5.1 U, and those of ice values ranged from 0.2 to 0.3 U. Inhibitor concentrations were 45 μM anti-Vph1p Fab and 45 μM nonimmune Fab.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Inhibition by antibodies to Vph1p. (A) Different concentrations of affinity-purified antibodies to Vph1p (from goat) were added to a standard fusion reaction without cytosol. The reaction was started after preincubation for 10 min on ice by adding ATP. Fusion was assayed after 70 min at 27°C. (B) Nonimmune antibodies do not interfere with vacuole fusion. Standard fusion reactions were run with 20 μM IgG purified from nonimmune or anti-Vph1p sera from goats, or with control buffer only, and assayed after 70 min at 27°C (n = 6). (C) Inhibition of fusion by Fab fragments of antibodies to Vph1p. Standard fusion reactions were incubated with Fab fragments derived either from nonimmune antibodies or from antibodies to Vph1p. Fusion was assayed as in the legend to Fig. 2 (n = 3). Fusion activities of the control samples (asterisk) ranged from 3.2 to 5.1 U, and those of ice values ranged from 0.2 to 0.3 U. Inhibitor concentrations were 45 μM anti-Vph1p Fab and 45 μM nonimmune Fab.
Mentions: We sought to specifically interfere with the fusion relevant aspect of V0. Available low molecular weight V-ATPase inhibitors affect only the proton translocation function of the V-ATPase, (e.g., concanamycin A) and/or are slowly reacting and of unsatisfactory specificity (e.g., dicyclohexylcarbodiimide). We raised antibodies to V0 subunits in order to generate highly specific agents that might interfere with V0 function and be suitable for analysis of the fusion reaction. We immunized goats with the recombinantly expressed and purified NH2-terminal cytosolic part of Vph1p and prepared affinity-purified polyclonal antibodies from the sera. If titrated into in vitro fusion reactions, these antibodies inhibited fusion in a concentration-dependent manner (IC50= 6 μM; Fig. 5 A). Nonimmune antibodies had no effect at comparable concentrations (Fig. 5 B). To exclude potential interference due to the divalent nature of whole IgG, we also prepared monovalent Fab fragments from these antibodies. They also inhibited fusion at concentrations comparable to those observed for whole IgGs (Fig. 5 C), matching our general experience for many other fusion relevant components: the inhibitory potency of whole IgGs and Fab fragments is not significantly different (Haas et al., 1995; Haas and Wickner, 1996; Mayer and Wickner, 1997; Nichols et al., 1997).

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