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J. Biol. Chem., Vol. 283, Issue 24, 16950-16959, June 13, 2008

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Functional Surfaces on the p35/ARPC2 Subunit of Arp2/3 Complex Required for Cell Growth, Actin Nucleation, and Endocytosis^*

Karen M. Daugherty and Bruce L. Goode¹

From the Departments of Biochemistry and Biology, Brandeis University and the Rosenstiel Basic Medical Sciences Center, Waltham, Massachusetts 02454

The Arp2/3 complex is comprised of seven evolutionarily conserved subunits and upon activation by WASp or another nucleation promoting factor nucleates the formation of actin filaments. These events are critical for driving a wide range of cellular processes, including motility, endocytosis, and intracellular trafficking. However, an in depth understanding of the Arp2/3 complex activation and nucleation mechanism is still lacking. Here, we used a mutagenesis approach in Saccharomyces cerevisiae to dissect the structural and functional roles of the p35/ARPC2 subunit. Using integrated alleles that target conserved and solvent-exposed residues, we identified surfaces on p35/ARPC2 required for cell growth, actin organization, and endocytosis. In parallel, we purified the mutant Arp2/3 complexes and compared their actin assembly activities both in the presence and in the absence of WASp. The majority of alleles with defects mapped to one face of p35/ARPC2, where there was a close correlation between loss of actin nucleation and endocytosis. A second site required for nucleation and endocytosis was identified near the contact surface between p35/ARPC2 and p19/ARPC4. A third site was identified at a more distal conserved surface, which was critical for endocytosis but not nucleation. These findings pinpoint the key surfaces on p35/ARPC2 required for Arp2/3 complex-mediated actin assembly and cellular function and provide a higher resolution view of Arp2/3 structure and mechanism.

Received for publication, January 30, 2008 , and in revised form, March 27, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM63691 (to B. G.). 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 Figs. S1-S4.

¹ To whom correspondence should be addressed: Dept. of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South St., Waltham, MA 02454. Tel.: 781-736-2464; Fax: 781-736-2405; E-mail: goode{at}brandeis.edu.

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