No abstract

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N-and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers.Remodeling of cell shape during cell motility, cell division, and cell morphogenesis requires not only the rapid assembly of new actin filaments but also the coordinated disassembly of older filaments. Dynamic turnover provides cells with the plasticity necessary to remodel actin networks rapidly in response to cues, and replenishes the pool of assembly competent ATP-bound actin monomers available for new growth. Although major advancements have been made in determining the mechanisms that promote actin assembly (1, 2), comparatively little is known about the mechanisms governing actin disassembly and turnover. The rate-limiting step in filament disassembly is dissociation of subunits from filament ends (3). actin depolymerizing factor/cofilin (referred to herein as cofilin) accelerates this step by severing and depolymerizing older (ADP-rich) actin filaments (4 -7). Cofilin remains bound to dissociated ADP-actin monomers and strongly inhibits nucleotide exchange (ATP for ADP) on monomeric actin (8). This leads to an accumulation of cofilin-bound ADP-actin monomers and depletes available cofilin and ATP-actin monomers. For this reason, cells require a mechanism for rapidly displacing cofilin from ADP-G-actin to maintain rapid actin turnover.Recent studies suggest that this function is performed by Srv2/cyclase-associated protein (CAP), 2 which here we refer to as Srv2 (9, 10). Srv2 is expressed ubiquitously in all animal, plant, and fungal or...

Profilin and cyclase-associated protein (CAP, known in yeast as Srv2) are ubiquitous and abundant actin monomer-binding proteins. Profilin catalyses the nucleotide exchange on actin monomers and promotes their addition to filament barbed ends. Srv2/CAP recycles newly depolymerized actin monomers from ADF/cofilin for subsequent rounds of polymerization. Srv2/CAP also harbors two proline-rich motifs and has been suggested to interact with profilin. However, the mechanism and biological role of the possible profilin-Srv2/CAP interaction has not been investigated. Here, we show that Saccharomyces cerevisiae Srv2 and profilin interact directly (KD ∼1.3 μM) and demonstrate that a specific proline-rich motif in Srv2 mediates this interaction in vitro and in vivo. ADP-actin monomers and profilin do not interfere with each other's binding to Srv2, suggesting that these three proteins can form a ternary complex. Genetic and cell biological analyses on an Srv2 allele (srv2-201) defective in binding profilin reveals that a direct interaction with profilin is not essential for Srv2 cellular function. However, srv2-201 causes a moderate increase in cell size and partially suppresses the cell growth and actin organization defects of an actin binding mutant profilin (pfy1-4). Together these data suggest that Srv2 is an important physiological interaction partner of profilin.