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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Time course of inhibition by LPC. 10× standard fusion reactions were started. 10 min before the indicated time points, 30-μl aliquots were supplemented with 1.5 μl of the indicated inhibitors or of buffer and placed on ice for 10 min. At the indicated time points, the samples were returned to 27°C and incubated until the end of the total reaction period of 70 min. One sample was kept on ice for each time point. The means of four independent experiments normalized to the controls are shown with SD. Fusion activity varied between 3.52 and 5.5 U. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 650 μM LPC-12, and 140 μM LPC-14.
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fig2: Time course of inhibition by LPC. 10× standard fusion reactions were started. 10 min before the indicated time points, 30-μl aliquots were supplemented with 1.5 μl of the indicated inhibitors or of buffer and placed on ice for 10 min. At the indicated time points, the samples were returned to 27°C and incubated until the end of the total reaction period of 70 min. One sample was kept on ice for each time point. The means of four independent experiments normalized to the controls are shown with SD. Fusion activity varied between 3.52 and 5.5 U. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 650 μM LPC-12, and 140 μM LPC-14.

Mentions: The latest reaction phase blocked by an inhibitor can be inferred from the kinetics with which the reaction becomes insensitive to the inhibitor. In such an experiment, a fusion reaction is incubated at 27°C. At certain time points, aliquots are removed, supplemented either with buffer (control; no change in the final fusion activity should be observed) or with inhibitor, and incubated further at 27°C until the end of a standard 70-min reaction period. Progression of an uninhibited reaction is monitored by transferring one aliquot to 0°C at every time point. This stops the reaction. The ice curve is indicative of fusion pore opening because there is no significant delay between contents mixing and the processing of the reporter pro-ALP (Merz and Wickner, 2004a). The reaction became insensitive to antibody inhibition of the priming ATPase Sec18p/NSF within 15 min (Mayer et al., 1996; Fig. 2). Gdi1p (guanine nucleotide dissociation inhibitor), which binds and partially extracts the Rab-GTPase Ypt7p from the membrane (Mayer and Wickner, 1997) and thereby blocks docking, inhibited up to 30 min. Inhibitors blocking the reaction between docking and fusion pore opening, such as the chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and guanosine-5′-(γ-thio)triphosphate (GTPγS), yield inhibition curves between those of Gdi1p and ice (Mayer et al., 1996; Peters and Mayer, 1998; Peters et al., 1999; Reese et al., 2005). LPC-14 and -12 showed such inhibition kinetics. The time courses for both inhibitors overlapped, suggesting that they affect the same late step of the reaction and act by the same mechanism. Because LPCs inhibit other fusion systems at the lipid-mixing stage (Chernomordik et al., 1998; Melikyan et al., 2000), this result suggests that lipid mixing is kinetically distinguishable from docking and significantly precedes contents mixing.


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

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

Time course of inhibition by LPC. 10× standard fusion reactions were started. 10 min before the indicated time points, 30-μl aliquots were supplemented with 1.5 μl of the indicated inhibitors or of buffer and placed on ice for 10 min. At the indicated time points, the samples were returned to 27°C and incubated until the end of the total reaction period of 70 min. One sample was kept on ice for each time point. The means of four independent experiments normalized to the controls are shown with SD. Fusion activity varied between 3.52 and 5.5 U. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 650 μM LPC-12, and 140 μM LPC-14.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Time course of inhibition by LPC. 10× standard fusion reactions were started. 10 min before the indicated time points, 30-μl aliquots were supplemented with 1.5 μl of the indicated inhibitors or of buffer and placed on ice for 10 min. At the indicated time points, the samples were returned to 27°C and incubated until the end of the total reaction period of 70 min. One sample was kept on ice for each time point. The means of four independent experiments normalized to the controls are shown with SD. Fusion activity varied between 3.52 and 5.5 U. The following inhibitors were used: 0.3 μM anti-Sec18p antibody, 1 μM Gdi1p, 650 μM LPC-12, and 140 μM LPC-14.
Mentions: The latest reaction phase blocked by an inhibitor can be inferred from the kinetics with which the reaction becomes insensitive to the inhibitor. In such an experiment, a fusion reaction is incubated at 27°C. At certain time points, aliquots are removed, supplemented either with buffer (control; no change in the final fusion activity should be observed) or with inhibitor, and incubated further at 27°C until the end of a standard 70-min reaction period. Progression of an uninhibited reaction is monitored by transferring one aliquot to 0°C at every time point. This stops the reaction. The ice curve is indicative of fusion pore opening because there is no significant delay between contents mixing and the processing of the reporter pro-ALP (Merz and Wickner, 2004a). The reaction became insensitive to antibody inhibition of the priming ATPase Sec18p/NSF within 15 min (Mayer et al., 1996; Fig. 2). Gdi1p (guanine nucleotide dissociation inhibitor), which binds and partially extracts the Rab-GTPase Ypt7p from the membrane (Mayer and Wickner, 1997) and thereby blocks docking, inhibited up to 30 min. Inhibitors blocking the reaction between docking and fusion pore opening, such as the chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and guanosine-5′-(γ-thio)triphosphate (GTPγS), yield inhibition curves between those of Gdi1p and ice (Mayer et al., 1996; Peters and Mayer, 1998; Peters et al., 1999; Reese et al., 2005). LPC-14 and -12 showed such inhibition kinetics. The time courses for both inhibitors overlapped, suggesting that they affect the same late step of the reaction and act by the same mechanism. Because LPCs inhibit other fusion systems at the lipid-mixing stage (Chernomordik et al., 1998; Melikyan et al., 2000), this result suggests that lipid mixing is kinetically distinguishable from docking and significantly precedes contents mixing.

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