Limits...
Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Reese C, Mayer A - J. Cell Biol. (2005)

Bottom Line: The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing.Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate.Pore opening was rate limiting for the reaction.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

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V0 trans-complexes in the presence of LPC. (A) 15× standard fusion reactions with cytosol were started using vacuoles from strains BJ Vph1-HA and -AU and the indicated inhibitors. One sample did not contain an ATP-regenerating system and was kept on ice. After 50 min, the reactions were stopped by chilling on ice and adding 1 ml PS buffer. Vacuoles were pelleted by centrifugation (21,000 g, 2 min, 4°C). Pellets were solubilized in 1.3 ml of precipitation buffer (150 mM KCl, 0.5 mM MnCl2, 1% [wt/vol] Triton X-100, and 1 mM PMSF in PS), shaken for 5 min at 4°C, and pelleted by centrifugation (21,000 g, 3 min, 4°C). To 1.1 ml of the solubilisate, 5 μl of monoclonal anti-HA antibody (raw ascites; 5–7 mg/ml) and 20 μl of protein G–Agarose was added. After 1 h of gentle shaking at 4°C, samples were washed with precipitation buffer, solubilized in SDS sample buffer, and analyzed by SDS-PAGE and Western blotting. (B) Stability of existing V0 trans-complexes against LPC-12. Fusion reactions were run as in A but without LPC-12. 40 min after the start of fusion, two of the samples (−10) were supplemented with LPC-12 in order to test the effect of LPC addition on V0 trans-complexes that had formed during the first 40 min. All samples were incubated for another 10 min at 27°C and then assayed for V0 trans-complexes as in A. The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 10 μM MED, and 4 mM GTPγS.
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fig6: V0 trans-complexes in the presence of LPC. (A) 15× standard fusion reactions with cytosol were started using vacuoles from strains BJ Vph1-HA and -AU and the indicated inhibitors. One sample did not contain an ATP-regenerating system and was kept on ice. After 50 min, the reactions were stopped by chilling on ice and adding 1 ml PS buffer. Vacuoles were pelleted by centrifugation (21,000 g, 2 min, 4°C). Pellets were solubilized in 1.3 ml of precipitation buffer (150 mM KCl, 0.5 mM MnCl2, 1% [wt/vol] Triton X-100, and 1 mM PMSF in PS), shaken for 5 min at 4°C, and pelleted by centrifugation (21,000 g, 3 min, 4°C). To 1.1 ml of the solubilisate, 5 μl of monoclonal anti-HA antibody (raw ascites; 5–7 mg/ml) and 20 μl of protein G–Agarose was added. After 1 h of gentle shaking at 4°C, samples were washed with precipitation buffer, solubilized in SDS sample buffer, and analyzed by SDS-PAGE and Western blotting. (B) Stability of existing V0 trans-complexes against LPC-12. Fusion reactions were run as in A but without LPC-12. 40 min after the start of fusion, two of the samples (−10) were supplemented with LPC-12 in order to test the effect of LPC addition on V0 trans-complexes that had formed during the first 40 min. All samples were incubated for another 10 min at 27°C and then assayed for V0 trans-complexes as in A. The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 10 μM MED, and 4 mM GTPγS.

Mentions: V0 sectors undergo a transformation in the final phase of vacuole fusion, which can be diagnosed by the association of V0 sectors from opposing membranes (Peters et al., 2001; Bayer et al., 2003). V0–V0 association preferentially affects a population of V0 sectors associated with the vacuolar t-SNARE Vam3p. It is a diagnostic criterion linked to the fusion cascade because it depends on vacuole priming and docking and is sensitive to the postdocking inhibitor BAPTA but insensitive to the latest acting inhibitor of vacuole fusion, GTPγS. Lipid transition mapped to the same stage as V0 transformation, i.e., it was insensitive to GTPγS but sensitive to BAPTA (Reese et al., 2005). This suggests that V0 transformation coincides with the induction of lipid flow. If so, changing lipid bilayer conformation by LPC might in turn influence V0 transformation. We tested the formation of V0 trans-complexes in the presence of LPCs or MED (Fig. 6). Vacuoles were prepared from two strains carrying either an HA or an AU tag on the V0 subunit Vph1p. The membranes were incubated under fusion conditions in the presence of various inhibitors. They were then solubilized, and Vph1p-HA was precipitated with a monoclonal anti-HA antibody. Vph1p-AU that was coimmunoprecipitated with Vph1p-HA was detected by Western blotting against the AU tag. Without ATP, a condition preventing priming and docking, no Vph1p trans-association was detected (Fig. 6 A). With ATP, Vph1p-AU coimmunoprecipitated with Vph1p-HA, indicating a stable association of the proteins. Gdi1p, the docking inhibitor that prevents trans-SNARE pairing, suppressed the formation of these V0–V0 complexes as completely as MED. In contrast, GTPγS, which inhibits after lipid mixing, permitted formation of V0 complexes, although it reduced fusion by >95%. In line with previous observations (Peters et al., 2001), this confirmed that the V0–V0 complexes observed arose from trans-association of V0–V0 sectors from two fusion partners before completion of fusion. LPC prevented V0 trans-association (Fig. 6 A). We also checked whether LPC destabilized an existing trans-association of V0. To this end, we ran fusion reactions for 40 min in the presence of GTPγS to generate V0 trans-complexes. LPC was added and, after 10 min of further incubation, the vacuoles were solubilized and assayed for V0 trans-complexes. The association of preformed V0 trans-complexes was only slightly affected by subsequent addition of LPCs (Fig. 6 B), demonstrating that LPCs do not destabilize V0 trans-complexes but interfere with their formation.


Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Reese C, Mayer A - J. Cell Biol. (2005)

V0 trans-complexes in the presence of LPC. (A) 15× standard fusion reactions with cytosol were started using vacuoles from strains BJ Vph1-HA and -AU and the indicated inhibitors. One sample did not contain an ATP-regenerating system and was kept on ice. After 50 min, the reactions were stopped by chilling on ice and adding 1 ml PS buffer. Vacuoles were pelleted by centrifugation (21,000 g, 2 min, 4°C). Pellets were solubilized in 1.3 ml of precipitation buffer (150 mM KCl, 0.5 mM MnCl2, 1% [wt/vol] Triton X-100, and 1 mM PMSF in PS), shaken for 5 min at 4°C, and pelleted by centrifugation (21,000 g, 3 min, 4°C). To 1.1 ml of the solubilisate, 5 μl of monoclonal anti-HA antibody (raw ascites; 5–7 mg/ml) and 20 μl of protein G–Agarose was added. After 1 h of gentle shaking at 4°C, samples were washed with precipitation buffer, solubilized in SDS sample buffer, and analyzed by SDS-PAGE and Western blotting. (B) Stability of existing V0 trans-complexes against LPC-12. Fusion reactions were run as in A but without LPC-12. 40 min after the start of fusion, two of the samples (−10) were supplemented with LPC-12 in order to test the effect of LPC addition on V0 trans-complexes that had formed during the first 40 min. All samples were incubated for another 10 min at 27°C and then assayed for V0 trans-complexes as in A. The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 10 μM MED, and 4 mM GTPγS.
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Related In: Results  -  Collection

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fig6: V0 trans-complexes in the presence of LPC. (A) 15× standard fusion reactions with cytosol were started using vacuoles from strains BJ Vph1-HA and -AU and the indicated inhibitors. One sample did not contain an ATP-regenerating system and was kept on ice. After 50 min, the reactions were stopped by chilling on ice and adding 1 ml PS buffer. Vacuoles were pelleted by centrifugation (21,000 g, 2 min, 4°C). Pellets were solubilized in 1.3 ml of precipitation buffer (150 mM KCl, 0.5 mM MnCl2, 1% [wt/vol] Triton X-100, and 1 mM PMSF in PS), shaken for 5 min at 4°C, and pelleted by centrifugation (21,000 g, 3 min, 4°C). To 1.1 ml of the solubilisate, 5 μl of monoclonal anti-HA antibody (raw ascites; 5–7 mg/ml) and 20 μl of protein G–Agarose was added. After 1 h of gentle shaking at 4°C, samples were washed with precipitation buffer, solubilized in SDS sample buffer, and analyzed by SDS-PAGE and Western blotting. (B) Stability of existing V0 trans-complexes against LPC-12. Fusion reactions were run as in A but without LPC-12. 40 min after the start of fusion, two of the samples (−10) were supplemented with LPC-12 in order to test the effect of LPC addition on V0 trans-complexes that had formed during the first 40 min. All samples were incubated for another 10 min at 27°C and then assayed for V0 trans-complexes as in A. The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 10 μM MED, and 4 mM GTPγS.
Mentions: V0 sectors undergo a transformation in the final phase of vacuole fusion, which can be diagnosed by the association of V0 sectors from opposing membranes (Peters et al., 2001; Bayer et al., 2003). V0–V0 association preferentially affects a population of V0 sectors associated with the vacuolar t-SNARE Vam3p. It is a diagnostic criterion linked to the fusion cascade because it depends on vacuole priming and docking and is sensitive to the postdocking inhibitor BAPTA but insensitive to the latest acting inhibitor of vacuole fusion, GTPγS. Lipid transition mapped to the same stage as V0 transformation, i.e., it was insensitive to GTPγS but sensitive to BAPTA (Reese et al., 2005). This suggests that V0 transformation coincides with the induction of lipid flow. If so, changing lipid bilayer conformation by LPC might in turn influence V0 transformation. We tested the formation of V0 trans-complexes in the presence of LPCs or MED (Fig. 6). Vacuoles were prepared from two strains carrying either an HA or an AU tag on the V0 subunit Vph1p. The membranes were incubated under fusion conditions in the presence of various inhibitors. They were then solubilized, and Vph1p-HA was precipitated with a monoclonal anti-HA antibody. Vph1p-AU that was coimmunoprecipitated with Vph1p-HA was detected by Western blotting against the AU tag. Without ATP, a condition preventing priming and docking, no Vph1p trans-association was detected (Fig. 6 A). With ATP, Vph1p-AU coimmunoprecipitated with Vph1p-HA, indicating a stable association of the proteins. Gdi1p, the docking inhibitor that prevents trans-SNARE pairing, suppressed the formation of these V0–V0 complexes as completely as MED. In contrast, GTPγS, which inhibits after lipid mixing, permitted formation of V0 complexes, although it reduced fusion by >95%. In line with previous observations (Peters et al., 2001), this confirmed that the V0–V0 complexes observed arose from trans-association of V0–V0 sectors from two fusion partners before completion of fusion. LPC prevented V0 trans-association (Fig. 6 A). We also checked whether LPC destabilized an existing trans-association of V0. To this end, we ran fusion reactions for 40 min in the presence of GTPγS to generate V0 trans-complexes. LPC was added and, after 10 min of further incubation, the vacuoles were solubilized and assayed for V0 trans-complexes. The association of preformed V0 trans-complexes was only slightly affected by subsequent addition of LPCs (Fig. 6 B), demonstrating that LPCs do not destabilize V0 trans-complexes but interfere with their formation.

Bottom Line: The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing.Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate.Pore opening was rate limiting for the reaction.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

Show MeSH
Related in: MedlinePlus