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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The fusion block with LPC is reversible. A 20× standard fusion reaction was started in the presence of 450 μM LPC-12. After 40 min at 27°C, 800 μl PS buffer with 15% (wt/vol) Ficoll and 0.5 mg/ml fatty acid–free bovine serum albumin were added. 300 μl PS buffer with 4% (wt/vol) Ficoll and 400 μl PS buffer were layered on top. Vacuoles were floated by centrifugation (21,000 g, 5 min, 4°C), recovered from the 0%/4% interface, and assayed for protein content. The recovered vacuoles were used in new standard fusion reactions with the indicated inhibitors. After 80 min, fusion was assayed. Control fusion reactions that had not been blocked with LPC and released were run in parallel. Their fusion activity was determined after 80 min at 27°C. Four independent experiments were averaged. Ice values had been subtracted. For the blocked and released reactions, ice values varied from 0.58 to 0.97 U and fusion activities varied from 2.2 to 4.2 U. For the unblocked reactions, ice values varied from 0.2 to 0.35 U and control values varied from 2.5 to 4.2 U. The following inhibitors were used: 1 μM Gdi1p, 5 mM BAPTA, and 4 mM GTPγS.
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fig7: The fusion block with LPC is reversible. A 20× standard fusion reaction was started in the presence of 450 μM LPC-12. After 40 min at 27°C, 800 μl PS buffer with 15% (wt/vol) Ficoll and 0.5 mg/ml fatty acid–free bovine serum albumin were added. 300 μl PS buffer with 4% (wt/vol) Ficoll and 400 μl PS buffer were layered on top. Vacuoles were floated by centrifugation (21,000 g, 5 min, 4°C), recovered from the 0%/4% interface, and assayed for protein content. The recovered vacuoles were used in new standard fusion reactions with the indicated inhibitors. After 80 min, fusion was assayed. Control fusion reactions that had not been blocked with LPC and released were run in parallel. Their fusion activity was determined after 80 min at 27°C. Four independent experiments were averaged. Ice values had been subtracted. For the blocked and released reactions, ice values varied from 0.58 to 0.97 U and fusion activities varied from 2.2 to 4.2 U. For the unblocked reactions, ice values varied from 0.2 to 0.35 U and control values varied from 2.5 to 4.2 U. The following inhibitors were used: 1 μM Gdi1p, 5 mM BAPTA, and 4 mM GTPγS.

Mentions: Because LPCs reversibly incorporate into membranes, we tried to rescue the fusion block by removing LPCs again (Chernomordik et al., 1997; Melikyan et al., 2000). Fusion reactions were started in the presence of LPC. After 40 min, LPC was extracted from the membranes by floating the vacuoles through fatty acid–free bovine serum albumin. New fusion reactions were set up with these reisolated organelles and tested for their sensitivities toward inhibitors (Fig. 7). Good fusion activity was recovered after removing LPC. This shows that the vacuoles were not irreversibly damaged by LPC. Fusion after reversing the LPC block was insensitive to the docking inhibitor Gdi1p, as expected from the kinetic analysis (Fig. 2) and the docking assay (Fig. 4). The reaction stayed sensitive, however, to the postdocking inhibitors BAPTA and GTPγS. This indicates that the LPC block is reversible, that the intermediate accumulating in the presence of LPC is productive, and that the BAPTA- and GTPγS-sensitive steps cannot be passed before the LPC-sensitive stage.


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

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

The fusion block with LPC is reversible. A 20× standard fusion reaction was started in the presence of 450 μM LPC-12. After 40 min at 27°C, 800 μl PS buffer with 15% (wt/vol) Ficoll and 0.5 mg/ml fatty acid–free bovine serum albumin were added. 300 μl PS buffer with 4% (wt/vol) Ficoll and 400 μl PS buffer were layered on top. Vacuoles were floated by centrifugation (21,000 g, 5 min, 4°C), recovered from the 0%/4% interface, and assayed for protein content. The recovered vacuoles were used in new standard fusion reactions with the indicated inhibitors. After 80 min, fusion was assayed. Control fusion reactions that had not been blocked with LPC and released were run in parallel. Their fusion activity was determined after 80 min at 27°C. Four independent experiments were averaged. Ice values had been subtracted. For the blocked and released reactions, ice values varied from 0.58 to 0.97 U and fusion activities varied from 2.2 to 4.2 U. For the unblocked reactions, ice values varied from 0.2 to 0.35 U and control values varied from 2.5 to 4.2 U. The following inhibitors were used: 1 μM Gdi1p, 5 mM BAPTA, and 4 mM GTPγS.
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Related In: Results  -  Collection

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fig7: The fusion block with LPC is reversible. A 20× standard fusion reaction was started in the presence of 450 μM LPC-12. After 40 min at 27°C, 800 μl PS buffer with 15% (wt/vol) Ficoll and 0.5 mg/ml fatty acid–free bovine serum albumin were added. 300 μl PS buffer with 4% (wt/vol) Ficoll and 400 μl PS buffer were layered on top. Vacuoles were floated by centrifugation (21,000 g, 5 min, 4°C), recovered from the 0%/4% interface, and assayed for protein content. The recovered vacuoles were used in new standard fusion reactions with the indicated inhibitors. After 80 min, fusion was assayed. Control fusion reactions that had not been blocked with LPC and released were run in parallel. Their fusion activity was determined after 80 min at 27°C. Four independent experiments were averaged. Ice values had been subtracted. For the blocked and released reactions, ice values varied from 0.58 to 0.97 U and fusion activities varied from 2.2 to 4.2 U. For the unblocked reactions, ice values varied from 0.2 to 0.35 U and control values varied from 2.5 to 4.2 U. The following inhibitors were used: 1 μM Gdi1p, 5 mM BAPTA, and 4 mM GTPγS.
Mentions: Because LPCs reversibly incorporate into membranes, we tried to rescue the fusion block by removing LPCs again (Chernomordik et al., 1997; Melikyan et al., 2000). Fusion reactions were started in the presence of LPC. After 40 min, LPC was extracted from the membranes by floating the vacuoles through fatty acid–free bovine serum albumin. New fusion reactions were set up with these reisolated organelles and tested for their sensitivities toward inhibitors (Fig. 7). Good fusion activity was recovered after removing LPC. This shows that the vacuoles were not irreversibly damaged by LPC. Fusion after reversing the LPC block was insensitive to the docking inhibitor Gdi1p, as expected from the kinetic analysis (Fig. 2) and the docking assay (Fig. 4). The reaction stayed sensitive, however, to the postdocking inhibitors BAPTA and GTPγS. This indicates that the LPC block is reversible, that the intermediate accumulating in the presence of LPC is productive, and that the BAPTA- and GTPγS-sensitive steps cannot be passed before the LPC-sensitive stage.

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