AN ALPHA-ACTININ-PROFILIN CHIMERA WITH 2 ALTERNATIVELY OPERATING ACTIN-BINDING SITES

Studying the mode of interaction between actin and actin-binding prote ins, we constructed a chimaeric protein consisting of the sequence for bovine profilin I (P), to which the sequence for the actin-binding do main of Dictyostelium discoideum alpha-actinin (alpha AP-2) was fused N-terminally. The resulting hybrid clone was expressed in Escherichia coli, and the chimaeric protein, alpha A1-2P, purified by affinity chr omatography on poly-(L-proline) (PLP) columns and identified using spe cific antibodies. High resolution electron microscopy demonstrated tha t this protein consists of two discrete subdomains. In biochemical, vi scometric and electron microscopic analyses, we shelved that both modu les in this molecule are biologically active. The chimaera binds to po ly-(L-proline) and inhibits the polymerization of G-actin in KCI, whic h is consistent with the assumption that the profilin part is intact. Inhibition of actin polymerization in KCI was stronger than that of th e parental profilin, and the K-d value of its interaction with rabbit skeletal muscle actin, as determined by falling ball viscometry, was s maller (mean value 0.5 x 10(-6) M, as compared to 1.9 x 10(-6) M for b ovine profilin). In 2mM MgCl2, the actin polymerized rapidly, consiste nt with the interpretation that under these conditions the chimaera, l ike profilin? is less efficient as an actin-sequestering agent. In the presence of alpha A1-2P, the resulting filaments were decorated with particles projecting from the filament axis, We conclude that under th ese conditions the alpha A1-2 domain of alpha A1-2P is preferentially active, attaching the chimaeric particles laterally to the filaments. Hence, the parental modules combined in alpha A1-2P permit this molecu le to switch from a G-actin-to an F-actin-binding form.