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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LPC inhibits vacuole fusion. (A) Titration of four different LPCs. Standard fusion reactions were incubated at 27°C in the presence of the indicated inhibitors at increasing concentrations. One sample was kept on ice. After 70 min, fusion activity was assayed. Values were normalized to the controls without inhibitors, and ice values were subtracted. The means of five independent experiments are shown with SD. Fusion activity in the positive controls varied from 2.29 to 5.45 U. (B) LPC and Vam7p in vacuole fusion. Standard fusion reactions were supplemented with 420 μM LPC-12 and 300 nM of purified recombinant Vam7p (rVam7) as indicated and incubated on ice for 10 min. An ATP-regenerating system or buffer was added, and fusion was started by incubating samples at 27°C. After 70 min, fusion activity was determined. The means ± SD of four independent experiments is shown.
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fig1: LPC inhibits vacuole fusion. (A) Titration of four different LPCs. Standard fusion reactions were incubated at 27°C in the presence of the indicated inhibitors at increasing concentrations. One sample was kept on ice. After 70 min, fusion activity was assayed. Values were normalized to the controls without inhibitors, and ice values were subtracted. The means of five independent experiments are shown with SD. Fusion activity in the positive controls varied from 2.29 to 5.45 U. (B) LPC and Vam7p in vacuole fusion. Standard fusion reactions were supplemented with 420 μM LPC-12 and 300 nM of purified recombinant Vam7p (rVam7) as indicated and incubated on ice for 10 min. An ATP-regenerating system or buffer was added, and fusion was started by incubating samples at 27°C. After 70 min, fusion activity was determined. The means ± SD of four independent experiments is shown.

Mentions: We investigated the effects of LPC on vacuole fusion by titrating LPCs of different acyl chain lengths into fusion reactions. LPCs spontaneously incorporate into lipid bilayers. They lack the fatty acid at the sn-C2 position of the glycerol backbone, conferring an inverted cone shape. At the C1 position, different fatty acids can be attached via ester linkages. We tested 1-caprylyl, 1-capryl, 1-lauroyl, and 1-myristoyl LPCs, carrying saturated fatty acids with 8, 10, 12, or 14 carbon atoms, respectively. All LPCs inhibited the fusion of yeast vacuoles (Fig. 1 A). The IC50 values increased from 30 μM for LPC-14 (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine) to 3 mM for LPC-8 (1-caprylyl-2-hydroxy-sn-glycero-3-phosphocholine), depending on the hydrocarbon chain length of the respective LPC. A similar trend was reported for influenza HA fusion (Chernomordik et al., 1997). This suggests that LPCs inhibit vacuole fusion and viral fusion by the same principle. Because the proteins involved in both reactions are very different, LPCs most likely inhibit via a structural influence on the lipid bilayer rather than a deleterious effect on a fusion protein.


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

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

LPC inhibits vacuole fusion. (A) Titration of four different LPCs. Standard fusion reactions were incubated at 27°C in the presence of the indicated inhibitors at increasing concentrations. One sample was kept on ice. After 70 min, fusion activity was assayed. Values were normalized to the controls without inhibitors, and ice values were subtracted. The means of five independent experiments are shown with SD. Fusion activity in the positive controls varied from 2.29 to 5.45 U. (B) LPC and Vam7p in vacuole fusion. Standard fusion reactions were supplemented with 420 μM LPC-12 and 300 nM of purified recombinant Vam7p (rVam7) as indicated and incubated on ice for 10 min. An ATP-regenerating system or buffer was added, and fusion was started by incubating samples at 27°C. After 70 min, fusion activity was determined. The means ± SD of four independent experiments is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171322&req=5

fig1: LPC inhibits vacuole fusion. (A) Titration of four different LPCs. Standard fusion reactions were incubated at 27°C in the presence of the indicated inhibitors at increasing concentrations. One sample was kept on ice. After 70 min, fusion activity was assayed. Values were normalized to the controls without inhibitors, and ice values were subtracted. The means of five independent experiments are shown with SD. Fusion activity in the positive controls varied from 2.29 to 5.45 U. (B) LPC and Vam7p in vacuole fusion. Standard fusion reactions were supplemented with 420 μM LPC-12 and 300 nM of purified recombinant Vam7p (rVam7) as indicated and incubated on ice for 10 min. An ATP-regenerating system or buffer was added, and fusion was started by incubating samples at 27°C. After 70 min, fusion activity was determined. The means ± SD of four independent experiments is shown.
Mentions: We investigated the effects of LPC on vacuole fusion by titrating LPCs of different acyl chain lengths into fusion reactions. LPCs spontaneously incorporate into lipid bilayers. They lack the fatty acid at the sn-C2 position of the glycerol backbone, conferring an inverted cone shape. At the C1 position, different fatty acids can be attached via ester linkages. We tested 1-caprylyl, 1-capryl, 1-lauroyl, and 1-myristoyl LPCs, carrying saturated fatty acids with 8, 10, 12, or 14 carbon atoms, respectively. All LPCs inhibited the fusion of yeast vacuoles (Fig. 1 A). The IC50 values increased from 30 μM for LPC-14 (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine) to 3 mM for LPC-8 (1-caprylyl-2-hydroxy-sn-glycero-3-phosphocholine), depending on the hydrocarbon chain length of the respective LPC. A similar trend was reported for influenza HA fusion (Chernomordik et al., 1997). This suggests that LPCs inhibit vacuole fusion and viral fusion by the same principle. Because the proteins involved in both reactions are very different, LPCs most likely inhibit via a structural influence on the lipid bilayer rather than a deleterious effect on a fusion protein.

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