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

Abstract

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal ␤-propeller and a secondary actin binding site lies in a comparable location on its C-terminal ␤-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding-and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N-and C-terminal propeller domains.

The flare Gene, Which Encodes the AIP1 Protein of Drosophila, Functions to Regulate F-Actin Disassembly in Pupal Epidermal Cells

“…Homologs of AIP1 are found in all animals, in plants, fungi, and other eukaryotes, and the protein is conserved throughout its length (supplemental Figure S1 at http:/ /www.genetics.org/supplemental/). AIP1 has been extensively studied in yeast and worms Rodal et al 1999Rodal et al , 2005Ono 2001;Mohri and Ono 2003;Voegtli et al 2003;Mohri et al 2004Mohri et al , 2006Ono et al 2004;Clark et al 2006;Okada et al 2006).…”

“…promote actin filament disassembly (Mohri and Ono 2003;Mohri et al 2004Mohri et al , 2006Ono et al 2004;Rodal et al 2005;Brieher et al 2006;Clark et al 2006;Okada et al 2006). In vivo evidence for this has been obtained in yeast (Okada et al 2006).…”

“…In yeast, animals, and plants the actin interacting protein 1 (AIP1) facilitates the ability of cofilin to promote actin depolymerization (Okada et al 1999(Okada et al , 2006Balcer et al 2003;Mohri and Ono 2003;Ono 2003;Ketelaar et al 2004;Ono et al 2004;Paavilainen et al 2004;Rodal et al 2005;Clark et al 2006). AIP1 has been found to bind directly to both actin and cofilin, but it is not thought that it has any actin depolymerizing activity on its own (Ono 2003;Ono et al 2004;Rodal et al 2005;Clark et al 2006;Okada et al 2006).…”

“…AIP1 has been found to bind directly to both actin and cofilin, but it is not thought that it has any actin depolymerizing activity on its own (Ono 2003;Ono et al 2004;Rodal et al 2005;Clark et al 2006;Okada et al 2006). In yeast cofilin is an essential gene, while AIP1 is not.…”

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).…”