Identification of Functional Residues on Caenorhabditis elegans Actin-interacting Protein 1 (UNC-78) for Disassembly of Actin Depolymerizing Factor/Cofilin-bound Actin Filaments

Abstract

Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin. AIP1 also caps the barbed end of ADF/cofilin-bound actin filament. However, the mechanism by which AIP1 interacts with ADF/cofilin and actin is not clearly understood. We determined the crystal structure of Caenorhabditis elegans AIP1 (UNC-78), which revealed 14 WD40 modules arranged in two seven-bladed ␤-propeller domains. The structure allowed for the mapping of conserved surface residues, and mutagenesis studies identified five residues that affected the ADF/cofilin-dependent actin filament disassembly activity. Mutations of these residues, which reside in blades 3 and 4 in the N-terminal propeller domain, had significant effects on the disassembly activity but did not alter the barbed end capping activity. These data support a model in which this conserved surface of AIP1 plays a direct role in enhancing fragmentation/depolymerization of ADF/cofilin-bound actin filaments but not in barbed end capping.Actin filaments are the core components of the highly dynamic actin cytoskeleton, which plays an essential role in a wide range of biological processes. Regulated fragmentation and depolymerization of actin filaments are important for disassembling specific cytoskeletal structures and maintaining high concentrations of monomeric actin, thereby enhancing actin filament turnover. Actin-depolymerizing factor (ADF) 1 / cofilin plays a central role in the disassembly process by accelerating monomer dissociation from the pointed ends and severing filaments (reviewed in Refs. 1-4).Actin-interacting protein 1 (AIP1) is a unique regulator of ADF/cofilin-mediated actin dynamics (reviewed in Ref. 5). The interaction between AIP1 and actin was originally detected by a yeast two-hybrid system (6). In vitro, AIP1 alone has negligible or very weak effects on actin dynamics, whereas, in the presence of ADF/cofilin, AIP1 enhances filament fragmentation (7-9). In addition, AIP1 has the ability to cap barbed ends and bind to the side of ADF/cofilin-bound filaments (10). Although the capping activity has been suggested to enhance filament fragmentation by preventing reannealing of the severed filaments (10, 11), direct microscopic observation of the effects of AIP1 on ADF/cofilin-bound filaments indicates that AIP1 actively disassembles ADF/cofilin-bound filaments, and simple barbed end capping by other capping agents does not enhance disassembly by ADF/cofilin (12). At present, the biological significance of the capping activity of AIP1 is not well understood.The mechanism by which AIP1 selectively interacts with ADF/cofilin-bound actin filaments is unknown. Actin residues in subdomains 3 and 4 participate in the two-hybrid interaction with yeast AIP1 (6). The AIP1-interacting residues in subdomain 3 are also required for interaction with cofilin (9), suggesting that AIP1 directly interacts with subdomain 4 of actin and indirectly with subdomain 3, which might be mediate...

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

“…All aip1p mutants were then cleaved from GST by a thrombin digest, with the exception of aip1-15p, which we were unable to cleave. Our findings as well as those documented by Mohri et al (2004) confirm that Aip1p-GST performs equally well as Aip1p in control disassembly assays. Each aip1p mutant was used in a severing assay, as described by Rodal et al (1999).…”

“…The inability of our aip1p mutants, with the exception of GST-aip1-15p, to show defects in a severing assay, although perplexing, is consistent with data from Mohri et al (2004). Using C. elegans Aip1p (UNC-78), they were able to isolate N-terminal but not C-terminal propeller mutants (of four tested) that were defective in actin filament disassembly.…”

“…The actin and cofilin binding footprints seem to indicate a simple model for lateral actin filament binding by Aip1p in which it straddles cofilin while potentially binding two adjacent actin monomers. Observations made by Mohri et al (2004) support this model, because they showed that a C. elegans aip1p (unc-78) mutant capable of binding but not disassembling cofilin-decorated actin filaments reaches its filament binding saturation at a 2:1 actinto-Aip1p ratio. Since actin has only one known Aip1p binding site and each Aip1p molecule has two actin binding sites, these data are consistent with the proposal that one molecule of Aip1p binds per two actin subunits within a filament.…”

“…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).…”

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…UNC-60B interacts with another actin severing protein, actin-interacting protein 1, coded for by the UNC-78 gene, that is also required for proper muscle thin filament assembly (Ono, 2001;Mohri and Ono, 2003;Mohri et al, 2006). This protein has two sevenblade propellers at each end of the protein that interact with the thin filament, and the UNC-60B actin depolymerizing factor/cofilin protein binds to the filament by wedging between the propellers (Ono, 2003;Mohri et al, 2004;Clark et al, 2006). Tropomyosin inhibits actin depolymerizing factor/cofilin activity (Ono and Ono, 2002;Yu and Ono, 2006).…”