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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Working model of late subreactions in vacuolar membrane fusion. SNAREs are initially found in cis-complexes in the same membrane composed of v- and t-SNAREs (light and dark gray, respectively). They are disassembled by the action of NSF/Sec18p and its cofactor α-SNAP/Sec17p during priming. Upon priming, Sec17p is released as soluble protein. The action of several proteins leads to docking and the formation of trans-SNARE complexes. LPC acts before the BAPTA-sensitive step. A late subreaction involves conformational changes in the V0 sector of the vacuolar ATPase, possibly resulting in lipid mixing. GTPγS inhibits fusion downstream of V0 and lipid mixing.
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fig9: Working model of late subreactions in vacuolar membrane fusion. SNAREs are initially found in cis-complexes in the same membrane composed of v- and t-SNAREs (light and dark gray, respectively). They are disassembled by the action of NSF/Sec18p and its cofactor α-SNAP/Sec17p during priming. Upon priming, Sec17p is released as soluble protein. The action of several proteins leads to docking and the formation of trans-SNARE complexes. LPC acts before the BAPTA-sensitive step. A late subreaction involves conformational changes in the V0 sector of the vacuolar ATPase, possibly resulting in lipid mixing. GTPγS inhibits fusion downstream of V0 and lipid mixing.

Mentions: In sum, our results provide a basis to kinetically map the requirement for lipid transition in the reaction sequence of vacuole fusion (Fig. 9). LPCs permit priming and docking. Lipid conformation is crucial for the transition from docking to fusion, i.e., to complete the BAPTA- and GTPγS-sensitive steps and to induce the conformational change of V0, which occurs between docking and full fusion.


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

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

Working model of late subreactions in vacuolar membrane fusion. SNAREs are initially found in cis-complexes in the same membrane composed of v- and t-SNAREs (light and dark gray, respectively). They are disassembled by the action of NSF/Sec18p and its cofactor α-SNAP/Sec17p during priming. Upon priming, Sec17p is released as soluble protein. The action of several proteins leads to docking and the formation of trans-SNARE complexes. LPC acts before the BAPTA-sensitive step. A late subreaction involves conformational changes in the V0 sector of the vacuolar ATPase, possibly resulting in lipid mixing. GTPγS inhibits fusion downstream of V0 and lipid mixing.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Working model of late subreactions in vacuolar membrane fusion. SNAREs are initially found in cis-complexes in the same membrane composed of v- and t-SNAREs (light and dark gray, respectively). They are disassembled by the action of NSF/Sec18p and its cofactor α-SNAP/Sec17p during priming. Upon priming, Sec17p is released as soluble protein. The action of several proteins leads to docking and the formation of trans-SNARE complexes. LPC acts before the BAPTA-sensitive step. A late subreaction involves conformational changes in the V0 sector of the vacuolar ATPase, possibly resulting in lipid mixing. GTPγS inhibits fusion downstream of V0 and lipid mixing.
Mentions: In sum, our results provide a basis to kinetically map the requirement for lipid transition in the reaction sequence of vacuole fusion (Fig. 9). LPCs permit priming and docking. Lipid conformation is crucial for the transition from docking to fusion, i.e., to complete the BAPTA- and GTPγS-sensitive steps and to induce the conformational change of V0, which occurs between docking and full 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