STRUCTURE, SUBUNIT TOPOLOGY, AND ACTIN-BINDING ACTIVITY OF THE ARP2 3COMPLEX FROM ACANTHAMOEBA/

The Arp2/3 complex, first isolated from Acanthamoeba castellani by aff inity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel prote ins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 n m by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry o f the subunits is 1:1:1:1:1:1:1, indicating the purified complex conta ins one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of simi lar to 13 x 10 nm, and mathematical models of such a shape and size ar e consistent with the measured hydrodynamic properties. Chemical cross -linking with a battery of cross-linkers of different spacer arm lengt hs and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of t he ameba, especially in linear structures, possibly actin filament bun dles, that lie perpendicular to the leading edge. Purified Arp2/3 comp lex binds actin filaments with a K-d Of 2.3 mu M and a stoichiometry o f approximately one complex molecule per actin monomer. In electron mi crographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structura l and topological models for the Arp2/3 complex and suggest that affin ity for actin filaments accounts for the localization of complex subun its to actin-rich regions of Acanthamoeba.