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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 4. — CAPS acceleration of fusion requires a functional PH domain. (A) CAPS PH domain fails to accelerate fusion but inhibits CAPS stimulation. Fusion reactions with 5 mol% PI 4,5-P₂–containing acceptor liposomes were conducted in the absence (PH −CAPS) or presence (PH +CAPS) of 1 μM CAPS, and the indicated concentrations of a CAPS GST-PH domain fusion protein. For clarity, points were selected and plotted every 7.5 min. Exponential fits of time courses are shown. (B) Phosphoinositide binding characteristics of CAPS PH domain. Indicated amounts of CAPS GST-PH domain fusion protein (either wild-type PH or mutant PH^DEE) immobilized on glutathione agarose beads were incubated with liposomes containing PI 4-P, PI 4,5-P₂, or PI 3,4-P₂ as described in Materials and methods. (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. Rate constants were determined by exponential fit to time courses in the absence or presence of 1 μM CAPS. Mean ± SEM values are shown for three independent experiments. (D) The CAPS PH domain mutant R558D/K560E/K561E with impaired phosphoinositide binding fails to accelerate fusion. Fusion reactions with 10 mol% PI 4,5-P₂–containing acceptor liposomes were conducted in the absence or presence of 1 μM wild-type CAPS or mutant CAPS (CAPS PH^DEE).