INTERACTION OF CAP-Z WITH ACTIN - THE NH2-TERMINAL DOMAINS OF THE ALPHA-1 AND BETA-SUBUNITS ARE NOT REQUIRED FOR ACTIN CAPPING, AND ALPHA-1-BETA AND ALPHA-2-BETA HETERODIMERS BIND DIFFERENTIALLY TO ACTIN

Cap Z is a widely distributed, highly conserved, heterodimeric protein that binds to the barbed ends of actin filaments, but does not sever filaments. In chicken, two variant cDNAs (alpha 1 and alpha 2) encodin g proteins homologous to the alpha subunit of Cap Z have been describe d versus one for the beta subunit. To establish the effect of each sub unit and of the two potential heterodimers (alpha 1 beta and alpha 2 b eta) on actin and to explore the functional domains of the proteins, R NA transcripts derived from these cDNAs were studied using in vitro tr anslation Sequential deletion mutants at the carboxyl and amino termin i of the alpha subunit and at the amino terminus of the beta subunit w ere constructed, and the ability of each mutant to recombine with its heterologous subunit was assessed by gel filtration. The interaction o f the individual subunits, heterodimers, and mutants with actin was st udied using cosedimentation and quantitative binding assays. This stud y demonstrates that 1) both alpha 1 beta and alpha 2 beta heterodimers assemble in vitro after translation and bind selectively to the barbe d ends of actin filaments; 2) the affinity of alpha 1 beta heterodimer s for actin (K-D = 1.6 x 10(-10) M) is similar to 4-fold higher than t hat of alpha 2 beta heterodimers (K-D = 6.3 x 10(-10) M); 3) the amino -terminal 40% of the alpha 1 subunit (amino acids 1-115) and the amino -terminal 31% of the beta subunit (amino acids 1-86) are not required for high affinity binding of Cap Z to the barbed ends of actin filamen ts; and 4) the carboxyl-terminal 55 amino acids of the alpha subunit a ppear to be required for binding to actin, as are the carboxyl-termina l 15 amino acids of the beta subunit.