Home | Help | Feedback | Subscriptions | Archive | Search | Early Release Articles

This Article Full Text PDF (Full Text) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by James, D. J. Articles by Martin, T. F.J. PubMed PubMed Citation Articles by James, D. J. Articles by Martin, T. F.J. Social Bookmarking                      What's this?

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/).

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

Home | Help | Feedback | Subscriptions | Archive | Search | Early Release Articles