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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Lipid mixing in the presence of LPC and MED. (A and C) Fusion reactions with vacuoles containing rhodamine-labeled lipids were set up. After addition of unlabeled acceptor vacuoles, fusion-dependent dilution of the lipids was monitored via the increase in relative fluorescence. Reactions were run in the presence of control buffer or of LPC as indicated. (B and D) Content mixing was assayed from identical samples run in parallel.
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fig5: Lipid mixing in the presence of LPC and MED. (A and C) Fusion reactions with vacuoles containing rhodamine-labeled lipids were set up. After addition of unlabeled acceptor vacuoles, fusion-dependent dilution of the lipids was monitored via the increase in relative fluorescence. Reactions were run in the presence of control buffer or of LPC as indicated. (B and D) Content mixing was assayed from identical samples run in parallel.

Mentions: To determine whether LPC inhibited merging of the membranes or fusion pore formation, we used a novel assay for vacuolar membrane fusion that detects the fusion-dependent flow of lipids from one fusion partner to the other (Reese et al., 2005). This assay is based on the concentration-dependent change in fluorescence intensity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-PE; rhodamine dye coupled to dipalmitoyl-phosphatidylethanolamine). We incorporated self-quenching concentrations (3 mole percent) of Rh-PE into the membranes of vacuoles isolated from one of our standard fusion strains (DKY6281) and mixed them with a sixfold excess of unlabeled vacuoles isolated from the other fusion strain (BJ3505). Upon fusion, the labeled phospholipids are diluted into the unlabeled BJ3505 membrane, which increases the relative fluorescence of rhodamine. Fusion reactions of identical composition can be tested for content mixing via the common vacuole fusion assay, which is based on the activation of pro-ALP in BJ3505 vacuoles by processing proteases provided by DKY6281 vacuoles. We titrated LPC-12 and MED into fusion reactions to test in parallel how LPC influences lipid and content mixing (Fig. 5). LPC-12 and MED inhibited both content- and lipid-mixing signals to comparable degrees. Thus, LPC and MED already suppress the lipid-mixing step of vacuolar membrane fusion.


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

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

Lipid mixing in the presence of LPC and MED. (A and C) Fusion reactions with vacuoles containing rhodamine-labeled lipids were set up. After addition of unlabeled acceptor vacuoles, fusion-dependent dilution of the lipids was monitored via the increase in relative fluorescence. Reactions were run in the presence of control buffer or of LPC as indicated. (B and D) Content mixing was assayed from identical samples run in parallel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171322&req=5

fig5: Lipid mixing in the presence of LPC and MED. (A and C) Fusion reactions with vacuoles containing rhodamine-labeled lipids were set up. After addition of unlabeled acceptor vacuoles, fusion-dependent dilution of the lipids was monitored via the increase in relative fluorescence. Reactions were run in the presence of control buffer or of LPC as indicated. (B and D) Content mixing was assayed from identical samples run in parallel.
Mentions: To determine whether LPC inhibited merging of the membranes or fusion pore formation, we used a novel assay for vacuolar membrane fusion that detects the fusion-dependent flow of lipids from one fusion partner to the other (Reese et al., 2005). This assay is based on the concentration-dependent change in fluorescence intensity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-PE; rhodamine dye coupled to dipalmitoyl-phosphatidylethanolamine). We incorporated self-quenching concentrations (3 mole percent) of Rh-PE into the membranes of vacuoles isolated from one of our standard fusion strains (DKY6281) and mixed them with a sixfold excess of unlabeled vacuoles isolated from the other fusion strain (BJ3505). Upon fusion, the labeled phospholipids are diluted into the unlabeled BJ3505 membrane, which increases the relative fluorescence of rhodamine. Fusion reactions of identical composition can be tested for content mixing via the common vacuole fusion assay, which is based on the activation of pro-ALP in BJ3505 vacuoles by processing proteases provided by DKY6281 vacuoles. We titrated LPC-12 and MED into fusion reactions to test in parallel how LPC influences lipid and content mixing (Fig. 5). LPC-12 and MED inhibited both content- and lipid-mixing signals to comparable degrees. Thus, LPC and MED already suppress the lipid-mixing step of vacuolar membrane fusion.

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