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. 2008 Jul 28;182(2):355–366. doi: 10.1083/jcb.200801056

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© 2008 James et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 3. — CAPS accelerates liposome fusion in a PI 4,5-P₂– and SNARE-dependent manner. (A) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 (indicated by t for t-SNAREs) in 90:10 mol% PC/PI 4,5-P₂ were incubated with donor liposomes with 100 copies of VAMP-2 in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS. Similar incubations with donor and acceptor liposomes in 85:15 mol% PC/PS in the absence or presence of 1 μM CAPS were conducted. NBD fluorescence in parallel reactions with Pf liposomes was used to correct all data. Mean values ± SEM for five independent experiments are shown. (B) Acceptor liposomes with 40 copies of syntaxin-1/SNAP-25 in 90:10 mol% PC/PI 4,5-P₂ were incubated with VAMP-2–containing donor liposomes in the absence or presence of 1 μM CAPS. Parallel incubations used botulinum neurotoxin B–treated donor liposomes incubated without or with 1 μM CAPS. (C) Syntaxin-1/SNAP-25 acceptor liposomes (indicated by t for t-SNAREs) with 10 mol% PI 4,5-P₂ or with 15% PS were incubated with VAMP-2 donor liposomes in the presence of indicated CAPS concentrations. Exponential fits of time courses were used to derive rate constants (k). Mean ± SEM values are shown for three independent experiments. (D) Rate constants were determined for fusion reactions without or with 1 μM CAPS in incubations that contained syntaxin-1/SNAP-25 acceptor liposomes with the indicated mole percentage PI 4,5-P₂. Data in inset show the percentage of maximal NBD fluorescence at 120 min in incubations without or with CAPS. Mean ± SEM values are shown for four independent experiments. (E) Extent of fusion in 120 min was determined in the absence or presence of 1 μM CAPS with donor VAMP-2 or acceptor syntaxin-1/SNAP-25 liposomes that contained 85:15 mol% PC/PS or 90:10 mol% PC/PI 4,5-P₂ as indicated. Mean ± SEM values are shown for three independent experiments (*, P < 0.01 for PC/PIP₂ compared with PC/PS; †, P = 0.05 compared with parallel incubations without CAPS).