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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Priming in the presence of LPC. 3× standard fusion reactions without ATP-regenerating system were preincubated with the indicated inhibitors (5 min, 0°C). The ATP-regenerating system or control buffer was added, and reactions were started by incubation at 27°C. The sample without ATP was kept on ice. After 15 min, the reactions were stopped by chilling on ice. 1 ml PS buffer with 150 mM KCl was added, and vacuoles were sedimented (21,000 g, 3 min, 4°C). 900 μl of the supernatant was transferred to a new reaction tube, TCA precipitated, and analyzed by SDS-PAGE and Western blotting. Vacuole pellets were resuspended in 200 μl of fusion buffer with 150 mM KCl, transferred to a new reaction tube, and centrifuged (21,000 g, 3 min, 4°C). Pellets were analyzed by SDS-PAGE and Western blotting. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 500 μM LPC-12, and 120 μM LPC-14.
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fig3: Priming in the presence of LPC. 3× standard fusion reactions without ATP-regenerating system were preincubated with the indicated inhibitors (5 min, 0°C). The ATP-regenerating system or control buffer was added, and reactions were started by incubation at 27°C. The sample without ATP was kept on ice. After 15 min, the reactions were stopped by chilling on ice. 1 ml PS buffer with 150 mM KCl was added, and vacuoles were sedimented (21,000 g, 3 min, 4°C). 900 μl of the supernatant was transferred to a new reaction tube, TCA precipitated, and analyzed by SDS-PAGE and Western blotting. Vacuole pellets were resuspended in 200 μl of fusion buffer with 150 mM KCl, transferred to a new reaction tube, and centrifuged (21,000 g, 3 min, 4°C). Pellets were analyzed by SDS-PAGE and Western blotting. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 500 μM LPC-12, and 120 μM LPC-14.

Mentions: The analysis in the preceding paragraph does not rule out the possibility that LPCs additionally inhibit earlier reaction steps. Therefore, we scored molecular marker events for priming and docking. Priming separates vacuolar cis-SNARE complexes through the action of Sec18p/NSF and Sec17p/α-SNAP (Ungermann et al., 1998a). As a consequence, Sec17p/α-SNAP is released from the vacuole into the supernatant (Mayer et al., 1996). We tested the influence of LPC-14 and -12 on Sec17p/α-SNAP release (Fig. 3). The vacuolar membrane protein ALP was blotted as a control for complete separation of membranes and supernatant. When vacuoles were incubated under fusion conditions without an ATP-regenerating system, Sec17p/α-SNAP remained associated with the vacuoles (Fig. 3). After addition of an ATP-regenerating system, Sec17p/ α-SNAP was released into the supernatant within 15 min. Antibodies to NSF/Sec18p inhibited this reaction, whereas the docking inhibitor Gdi1p did not influence it. Neither LPC-14 nor -12 blocked Sec17p release.


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

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

Priming in the presence of LPC. 3× standard fusion reactions without ATP-regenerating system were preincubated with the indicated inhibitors (5 min, 0°C). The ATP-regenerating system or control buffer was added, and reactions were started by incubation at 27°C. The sample without ATP was kept on ice. After 15 min, the reactions were stopped by chilling on ice. 1 ml PS buffer with 150 mM KCl was added, and vacuoles were sedimented (21,000 g, 3 min, 4°C). 900 μl of the supernatant was transferred to a new reaction tube, TCA precipitated, and analyzed by SDS-PAGE and Western blotting. Vacuole pellets were resuspended in 200 μl of fusion buffer with 150 mM KCl, transferred to a new reaction tube, and centrifuged (21,000 g, 3 min, 4°C). Pellets were analyzed by SDS-PAGE and Western blotting. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 500 μM LPC-12, and 120 μM LPC-14.
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getmorefigures.php?uid=PMC2171322&req=5

fig3: Priming in the presence of LPC. 3× standard fusion reactions without ATP-regenerating system were preincubated with the indicated inhibitors (5 min, 0°C). The ATP-regenerating system or control buffer was added, and reactions were started by incubation at 27°C. The sample without ATP was kept on ice. After 15 min, the reactions were stopped by chilling on ice. 1 ml PS buffer with 150 mM KCl was added, and vacuoles were sedimented (21,000 g, 3 min, 4°C). 900 μl of the supernatant was transferred to a new reaction tube, TCA precipitated, and analyzed by SDS-PAGE and Western blotting. Vacuole pellets were resuspended in 200 μl of fusion buffer with 150 mM KCl, transferred to a new reaction tube, and centrifuged (21,000 g, 3 min, 4°C). Pellets were analyzed by SDS-PAGE and Western blotting. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 500 μM LPC-12, and 120 μM LPC-14.
Mentions: The analysis in the preceding paragraph does not rule out the possibility that LPCs additionally inhibit earlier reaction steps. Therefore, we scored molecular marker events for priming and docking. Priming separates vacuolar cis-SNARE complexes through the action of Sec18p/NSF and Sec17p/α-SNAP (Ungermann et al., 1998a). As a consequence, Sec17p/α-SNAP is released from the vacuole into the supernatant (Mayer et al., 1996). We tested the influence of LPC-14 and -12 on Sec17p/α-SNAP release (Fig. 3). The vacuolar membrane protein ALP was blotted as a control for complete separation of membranes and supernatant. When vacuoles were incubated under fusion conditions without an ATP-regenerating system, Sec17p/α-SNAP remained associated with the vacuoles (Fig. 3). After addition of an ATP-regenerating system, Sec17p/ α-SNAP was released into the supernatant within 15 min. Antibodies to NSF/Sec18p inhibited this reaction, whereas the docking inhibitor Gdi1p did not influence it. Neither LPC-14 nor -12 blocked Sec17p release.

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