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.

Show MeSH

Related in: MedlinePlus

Fusion kinetics after fusion block. Vacuoles were preincubated under fusion conditions with 5 mM BAPTA, 4 mM GTPγS, 420 μM LPC-12, and 250 mM KCl or without ATP-regenerating system for 40 min. Vacuoles were then reisolated as in Fig. 7 and used to set up new fusion reactions without inhibitors. Aliquots were removed every 10 min and set on ice. After 80 min, fusion was assayed. Four independent experiments were averaged. Measured ALP activities varied from 0.4 to 0.6 U for the 0-min value and from 1.6 to 2.3 U for the 80-min value.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171322&req=5

fig8: Fusion kinetics after fusion block. Vacuoles were preincubated under fusion conditions with 5 mM BAPTA, 4 mM GTPγS, 420 μM LPC-12, and 250 mM KCl or without ATP-regenerating system for 40 min. Vacuoles were then reisolated as in Fig. 7 and used to set up new fusion reactions without inhibitors. Aliquots were removed every 10 min and set on ice. After 80 min, fusion was assayed. Four independent experiments were averaged. Measured ALP activities varied from 0.4 to 0.6 U for the 0-min value and from 1.6 to 2.3 U for the 80-min value.

Mentions: The availability of several reversible and late-acting inhibitors enabled us to compare the kinetics of vacuole fusion after release from these blocks. Should an early subreaction be rate limiting, a reaction arrested at a stage past this rate-limiting event should proceed significantly faster than a standard reaction. In contrast, if a late event in the reaction cascade is rate limiting, a reaction rescued from arrest before or at this rate-limiting step should run with a similar rate as an uninhibited reaction. We determined fusion kinetics after release from reversible blocks. Fusion was started in the presence of the inhibitors to allow the vacuoles to proceed up to the targeted reaction stage. Then the vacuoles were reisolated to remove the inhibitors and used in new fusion reactions. After restarting the reactions, aliquots were transferred to 0°C in 10-min intervals to stop further fusion and monitor the progress of the reaction. We compared the fusion rates after release from postdocking blocks with LPC, GTPγS, and BAPTA to those of reactions released from a high-salt block (250 mM KCl, which arrests fusion at docking; unpublished data) and to a reaction without inhibitor, in which fusion had been prevented by omission of ATP. MED was not used in this experiment because its inhibitory effect was not reversible by a simple washing of the membranes (unpublished data). The rates after recovery from all reversible blocks were similar to that of an uninhibited standard reaction (Fig. 8). It appears unlikely that incomplete removal of the inhibitors after the block or slow dissociation of the inhibitors from their targets could limit the fusion rate after rescue because the inhibitors are unrelated by chemical structure and by mode of action. Therefore, the rate-limiting step of vacuole fusion should lie at or beyond the stages sensitive to LPC, BAPTA, and GTPγS. Because docking and lipid flow between the fusion partners occurs in the presence of GTPγS (Reese et al., 2005), our data suggests that the rate-limiting step in the fusion pathway lies downstream of these events.


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

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

Fusion kinetics after fusion block. Vacuoles were preincubated under fusion conditions with 5 mM BAPTA, 4 mM GTPγS, 420 μM LPC-12, and 250 mM KCl or without ATP-regenerating system for 40 min. Vacuoles were then reisolated as in Fig. 7 and used to set up new fusion reactions without inhibitors. Aliquots were removed every 10 min and set on ice. After 80 min, fusion was assayed. Four independent experiments were averaged. Measured ALP activities varied from 0.4 to 0.6 U for the 0-min value and from 1.6 to 2.3 U for the 80-min value.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Fusion kinetics after fusion block. Vacuoles were preincubated under fusion conditions with 5 mM BAPTA, 4 mM GTPγS, 420 μM LPC-12, and 250 mM KCl or without ATP-regenerating system for 40 min. Vacuoles were then reisolated as in Fig. 7 and used to set up new fusion reactions without inhibitors. Aliquots were removed every 10 min and set on ice. After 80 min, fusion was assayed. Four independent experiments were averaged. Measured ALP activities varied from 0.4 to 0.6 U for the 0-min value and from 1.6 to 2.3 U for the 80-min value.
Mentions: The availability of several reversible and late-acting inhibitors enabled us to compare the kinetics of vacuole fusion after release from these blocks. Should an early subreaction be rate limiting, a reaction arrested at a stage past this rate-limiting event should proceed significantly faster than a standard reaction. In contrast, if a late event in the reaction cascade is rate limiting, a reaction rescued from arrest before or at this rate-limiting step should run with a similar rate as an uninhibited reaction. We determined fusion kinetics after release from reversible blocks. Fusion was started in the presence of the inhibitors to allow the vacuoles to proceed up to the targeted reaction stage. Then the vacuoles were reisolated to remove the inhibitors and used in new fusion reactions. After restarting the reactions, aliquots were transferred to 0°C in 10-min intervals to stop further fusion and monitor the progress of the reaction. We compared the fusion rates after release from postdocking blocks with LPC, GTPγS, and BAPTA to those of reactions released from a high-salt block (250 mM KCl, which arrests fusion at docking; unpublished data) and to a reaction without inhibitor, in which fusion had been prevented by omission of ATP. MED was not used in this experiment because its inhibitory effect was not reversible by a simple washing of the membranes (unpublished data). The rates after recovery from all reversible blocks were similar to that of an uninhibited standard reaction (Fig. 8). It appears unlikely that incomplete removal of the inhibitors after the block or slow dissociation of the inhibitors from their targets could limit the fusion rate after rescue because the inhibitors are unrelated by chemical structure and by mode of action. Therefore, the rate-limiting step of vacuole fusion should lie at or beyond the stages sensitive to LPC, BAPTA, and GTPγS. Because docking and lipid flow between the fusion partners occurs in the presence of GTPγS (Reese et al., 2005), our data suggests that the rate-limiting step in the fusion pathway lies downstream of these events.

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