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Published online
doi:10.1083/jcb.200801056
The Journal of Cell Biology, Vol. 182, No. 2, 355-366
The Rockefeller University Press, 0021-9525 $30.00
© James et al.
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Article

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion



Declan J. James, Chuenchanok Khodthong, Judith A. Kowalchyk, and Thomas F.J. Martin

Department of Biochemistry, University of Wisconsin, Madison, WI 53706

Correspondence to Thomas F.J. Martin: tfmartin{at}wisc.edu

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P2 as ~6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P2 soluble NSF attachment protein receptor (SNARE)–dependent liposome fusion is inhibited. Inhibition by PI 4,5-P2 likely results from its intrinsic positive curvature–promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P2, suggesting that syntaxin sequesters PI 4,5-P2 to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P2, we test a PI 4,5-P2–binding priming factor required for vesicle exocytosis. Ca2+-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P2 dependent. These results indicate that PI 4,5-P2 regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

Abbreviations used in this paper: CAPS, Ca2+-dependent activator protein for secretion; DOPC, 1,2-dioleoyl phosphatidylcholine; DOPS, 1,2-dioleoyl phosphatidylserine; LPC, lysophosphatidylcholine; NBD-PE, N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; Pf, protein-free; PH, pleckstrin homology; PI 4-P, phosphatidylinositol-4-monophosphate; PI 4,5-P2, phosphatidylinositol-4,5-bisphosphate; PS, phosphatidylserine; Rh-PE, N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine; SNAP-25, 25-kD synaptosome-associated protein; VAMP-2, vesicle-associated membrane protein 2.

© 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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