The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine othe r proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate t hat myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that cap ping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 r eveals a protein dominated by leucine-rich repeats and proline-rich sequenc es, and indicates that it is a homologue of Acan 125. Studies using p116 fu sion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding prot eins, as well as an acidic stretch, can activate Arp2/3-dependent actin nuc leation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dy namic astin-rich cellular extensions, including the leading edge of cells u ndergoing chemotactic migration, and dorsal, cup-like, macropinocytic exten sions. Cells lacking p116 exhibit a striking defect in the formation of the se macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results iden tify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indica te that the protein responsible for the formation of this complex is physio logically important, and suggest that previously reported myosin I mutant p henotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homo logues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.