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) encoding proteins homologous to the alpha subunit of Cap Z have been described versus one for the beta subunit. To establish the effect of each subunit and of the two potential heterodimers (alpha 1 beta and alpha 2 beta) on actin and to explore the functional domains of the proteins, RNA transcripts derived from these cDNAs were studied using in vitro translation. Sequential deletion mutants at the carboxyl and amino termini of the alpha subunit and at the amino terminus of the beta subunit were constructed, and the ability of each mutant to recombine with its heterologous subunit was assessed by gel filtration. The interaction of the individual subunits, heterodimers, and mutants with actin was studied using cosedimentation and quantitative binding assays. This study demonstrates that 1) both alpha 1 beta and alpha 2 beta heterodimers assemble in vitro after translation and bind selectively to the barbed ends of actin filaments; 2) the affinity of alpha 1 beta heterodimers for actin (KD = 1.6 x 10(-10) M) is approximately 4-fold higher than that of alpha 2 beta heterodimers (KD = 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 filaments; and 4) the carboxyl-terminal 55 amino acids of the alpha subunit appear to be required for binding to actin, as are the carboxyl-terminal 15 amino acids of the beta subunit.