Reconstitution and Dissection of the 600-kDa Srv2/CAP Complex

Abstract

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...

Structure and Function of a G-actin Sequestering Protein with a Vital Role in Malaria Oocyst Development inside the Mosquito Vector

“…In good agreement, CAP associates directly with G-actin (11) through its C-terminal domain (12,13). CAP binds preferentially to ADP-actin monomers (14) and also participates, together with actin and other actin-binding proteins, in high molecular mass complexes the functions of which are not yet fully understood (15,16).…”

“…3A). The N-terminal ␣-helical bundle domain of conventional CAPs, which has recently been demonstrated to contribute significantly to ADF-mediated F-actin turnover (16), is absent in parasite C-CAPs. In line with this evidence, our data suggest that the consensus sequence of C-CAP proteins of apicomplexan parasites supports only dimerization.…”

“…4B). Incidentally, the absence in yeast CAP/Srv2p of a small aromatic side chain (Phe or Tyr), which in our structure stack to each other at the dimer interface, may be compensated for by other dimer interactions, such as those of the yeast N-terminal CAP domain (9,16). The internal Cys/Ser side chain stack is shared with other ␤-helical proteins (41), but a prominent difference between the parasite and yeast C-CAP structures is the dipartite ladder observed in our CpC-CAP structure, with an N-terminal Cys/Ser stack and a C-terminal hydrophobic stack (supplemental Fig.…”

“…After the discovery of more complex actin-associated CAP functions the emerging cellular role of conventional CAPs is that of a scaffold that regulates diverse Fand G-actin-related activities. Given the several distinct modes of CAP-actin interactions proposed (15,16), it is currently unclear which residues in each C-CAP monomer contribute to actin binding, and at what stoichiometry. Parasite C-CAPs lack the N-terminal proline-rich extensions that, in conventional CAPs, recruit SH3-domaincontaining proteins (36) and profilin (14,37).…”

ATP-dependent regulation of actin monomer–filament equilibrium by cyclase-associated protein and ADF/cofilin

“…CAP has multiple domains with distinct functions. Studies on human CAP1 and yeast CAP (Srv2/CAP) show that the N-terminal helical-folded domain binds to ADF/cofilin-actin complex, and that the central Wiscott Aldrich syndrome protein homology 2 (WH2) domain and the C-terminal CAPs and X-linked retinitis pigmentosa 2 protein (CARP) domain [16] mediate nucleotide exchange [9, 12, 14, 15]. However, the mechanism by which CAP promotes actin turnover remains elusive.…”