Journal of Biological Chemistry
Volume 282, Issue 38, 21 September 2007, Pages 28014-28024
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Molecular Basis of Cell and Developmental Biology
Single Molecule Kinetic Analysis of Actin Filament Capping: POLYPHOSPHOINOSITIDES DO NOT DISSOCIATE CAPPING PROTEINS*

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We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 × 106 m-1 s-1 and dissociation of 0.0003 s-1 agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), PI(4,5)P2, and PI(3,4,5)P3, prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 × 105 m-1 s-1) and a faster dissociation rate constant (0.004 s-1). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.

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1

Present address: Dept. of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406.

*

This work was supported by National Institutes of Health Research Grant GM-26338 (to T. D. P.) and a Burroughs Wellcome Fund Career Award at the Scientific Interface (to J. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.