Figure 2.
Figure 2. PI 4,5-P2 inhibits SNARE-dependent liposome fusion. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (t-SNARE) in 90:10 mol% PC/PI 4,5-P2 or 85:15 mol% PC/PS were mixed with fluorescent PC/PS donor liposomes with 100 copies of VAMP-2. Parallel reactions with Pf acceptor liposomes at 90:10 mol% PC/PI 4,5-P2 or 85:15 mol% PC/PS were conducted. The kinetics of fusion are shown as a percentage of total NBD fluorescence after correction for Pf liposomes (see Materials and methods). Points represent mean values ± SEM for five independent experiments. (B) Acceptor liposomes reconstituted with syntaxin-1/SNAP-25 and the indicated mole percentage PI 4,5-P2 were mixed with donor liposomes at a 9:1 ratio (∼2 mM lipid) and NBD fluorescence was monitored. The extent of fusion at 120 min is expressed as a percentage of fusion relative to that observed using acceptor liposomes with 85:15 mol% PC/PS. Bars indicate mean values ± SEM for three independent experiments (*, P < 0.01). (C) Liposome fusion reactions were conducted with VAMP-2 donor liposomes incubated with syntaxin-1/SNAP-25 acceptor liposomes containing 5 mol% PI 4-P, PI 4,5-P2, or PI 3,4-P2 as indicated. The extent of fusion at 120 min is expressed as in B (**, P < 0.001 compared with PC/PS donor–acceptor liposomes). (D) Inhibition of fusion by inverted cone lipid LPC. Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 85:15 mol% PC/PS or 90:5 mol% PC/PI 4,5-P2 as indicated were incubated 5 min at 30°C without or with 0.2 mM LPC and mixed with donor liposomes to monitor fusion kinetics. Values (mean ± SEM; n = 6) of percentage of maximal NBD fluorescence at 120-min incubation times are shown (**, P < 0.001 compared with untreated PC/PS liposomes). (E) The extent of liposome fusion at 120 min is shown for reactions containing donor liposomes mixed with acceptor liposomes containing wild-type or mutant (K252,253A, K264,265A, and K252,253,264,265A) syntaxins in 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P2 as indicated. Extent of fusion is shown as a percentage relative to wild-type syntaxin in PC/PS liposomes (**, P < 0.001 compared with WT syntaxin containing PC/PS liposomes).

PI 4,5-P2 inhibits SNARE-dependent liposome fusion. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (t-SNARE) in 90:10 mol% PC/PI 4,5-P2 or 85:15 mol% PC/PS were mixed with fluorescent PC/PS donor liposomes with 100 copies of VAMP-2. Parallel reactions with Pf acceptor liposomes at 90:10 mol% PC/PI 4,5-P2 or 85:15 mol% PC/PS were conducted. The kinetics of fusion are shown as a percentage of total NBD fluorescence after correction for Pf liposomes (see Materials and methods). Points represent mean values ± SEM for five independent experiments. (B) Acceptor liposomes reconstituted with syntaxin-1/SNAP-25 and the indicated mole percentage PI 4,5-P2 were mixed with donor liposomes at a 9:1 ratio (∼2 mM lipid) and NBD fluorescence was monitored. The extent of fusion at 120 min is expressed as a percentage of fusion relative to that observed using acceptor liposomes with 85:15 mol% PC/PS. Bars indicate mean values ± SEM for three independent experiments (*, P < 0.01). (C) Liposome fusion reactions were conducted with VAMP-2 donor liposomes incubated with syntaxin-1/SNAP-25 acceptor liposomes containing 5 mol% PI 4-P, PI 4,5-P2, or PI 3,4-P2 as indicated. The extent of fusion at 120 min is expressed as in B (**, P < 0.001 compared with PC/PS donor–acceptor liposomes). (D) Inhibition of fusion by inverted cone lipid LPC. Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 85:15 mol% PC/PS or 90:5 mol% PC/PI 4,5-P2 as indicated were incubated 5 min at 30°C without or with 0.2 mM LPC and mixed with donor liposomes to monitor fusion kinetics. Values (mean ± SEM; n = 6) of percentage of maximal NBD fluorescence at 120-min incubation times are shown (**, P < 0.001 compared with untreated PC/PS liposomes). (E) The extent of liposome fusion at 120 min is shown for reactions containing donor liposomes mixed with acceptor liposomes containing wild-type or mutant (K252,253A, K264,265A, and K252,253,264,265A) syntaxins in 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P2 as indicated. Extent of fusion is shown as a percentage relative to wild-type syntaxin in PC/PS liposomes (**, P < 0.001 compared with WT syntaxin containing PC/PS liposomes).

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