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Vol. 15, Issue 11, 5158-5171, November 2004

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A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

Pieta K. Mattila ^* , Omar Quintero-Monzon  , Jamie Kugler , James B. Moseley , Steven C. Almo , Pekka Lappalainen ^*, and Bruce L. Goode  ^||

^* Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Helsinki 00014, Finland; Department of Biology and The Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, MA, 02454; and Departments of Biochemistry and Anatomy and Structural Biology, and Center for Synchrotron Biosciences, Albert Einstein College of Medicine, Bronx, NY 10461

Submitted June 2, 2004; Accepted August 24, 2004
Monitoring Editor: David Drubin

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (K_d 0.02 µM) compared with ATP-G-actin (K_d 1.9 µM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved -sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

The online version of this article contains supplementary material accessible through http://www.molbiolcell.org.

These authors contributed equally to this work.

^|| Corresponding author. E-mail address: goode{at}brandeis.edu.

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