Coordinated Regulation of Actin Filament Turnover by a High-Molecular-Weight Srv2/CAP Complex, Cofilin, Profilin, and Aip1

Abstract

We define two genetically and biochemically separable functions for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

“…As a whole, these findings show that both individual actin binding sites and the cofilin binding site are necessary for proper Aip1p activity in vivo. If Aip1p is truly a barbed-end capping protein as reported previously (Okada et al, 2002;Balcer et al, 2003), we would expect the aip1 mutants to have more serious synthetic defects with the cap⌬ alleles, because they would be expected to be redundant in function. Interestingly, the actin binding site-specific aip1 alleles are more defective with the rvs161/ 167 deletions than they are with the cap1/cap2 deletions, suggesting a greater functional involvement of Aip1p and possibly Aip1p-facilitated severing with the RVS proteins than with the Cap proteins.…”

“…The ability of Aip1p to enhance severing of cofilin-decorated actin filaments has been convincingly documented (Okada et al, 1999;Ono et al, 2004). In addition, some findings indicate that Aip1p is able to cap the barbed ends of actin filaments, preventing filament elongation and thus enhancing disassembly (Okada et al, 2002;Balcer et al, 2003). Previous actin filament elongation assays using a range of XAip1 concentrations combined with cofilin provided persuasive evidence that XAip1 may exert a barbedend capping activity (Okada et al, 2002).…”

“…We also intended to test our aip1p mutants using a biochemical actin filament-capping assay (Balcer et al, 2003). In this assay, F-actin is combined with profilin, cofilin, and Aip1p at a 20:40:4:1 ratio and allowed to incubate at room temperature for 20 min.…”

“…The biochemical activities of Aip1p from multiple sources (S. cerevisiae, X. laevis, and C. elegans) have been extensively tested in vitro (Okada et al, 1999(Okada et al, , 2002Rodal et al, 1999;Balcer et al, 2003;Mohri and Ono, 2003;Mohri et al, 2004;Ono et al, 2004). In actin pelleting assays, S. cerevisiae Aip1p very strongly induces the disassembly of cofilin-bound actin filaments, whereas C. elegans Aip1p (UNC-78) does so moderately, and X. laevis Aip1p (XAip1) does so very weakly (Okada et al, 1999;Rodal et al, 1999;Mohri and Ono, 2003).…”

“…Biochemical assays show that cofilin promotes depolymerization of actin filaments by accelerating pointed-end filament disassembly (Carlier et al, 1997;Lappalainen and Drubin, 1997) and by a severing activity that is very weak at physiological pH (Maciver et al, 1991;Ichetovkin et al, 2000). Aip1p dramatically enhances the depolymerization activity of cofilin-decorated actin filaments and is suspected to do so by assisting cofilin-induced severing and/or by capping the barbed ends of actin filaments (Okada et al, 1999;Rodal et al, 1999;Okada et al, 2002;Balcer et al, 2003;Mohri et al, 2004;Ono et al, 2004).…”