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J. Biol. Chem., Vol. 283, Issue 39, 26490-26498, September 26, 2008

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Structure and Biochemical Properties of Fission Yeast Arp2/3 Complex Lacking the Arp2 Subunit^*

From the Departments of Molecular, Cellular and Developmental Biology, Cell Biology, and Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103

Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2 Arp2/3 complex) from fission yeast. An x-ray crystal structure of the Arp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2 Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2 Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

Received for publication, April 3, 2008 , and in revised form, June 17, 2008.

The atomic coordinates and structure factors (code 3DWL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM066311. This work was also supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship GM074374-02. 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 text and Figs. S1–S4.

¹ To whom correspondence should be addressed: Tel.: 203-432-3194; Fax: 203-432-6161; E-mail: bradley.nolen{at}yale.edu.

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