Interaction of Cap Z with Actin: The NH₂-terminal domains of the α1 and β subunits are not required for actin capping, and α1β and α2β 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 (α1and α2) encoding proteins homologous to the a subunit of Cap Z have been described versus one for the β subunit. To establish the effect of each subunit and of the two potential heterodimers (α1β and α2β) 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 a subunit and at the amino terminus of the β 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 α1β and α2β heterodimers assemble in vitro after translation and bind selectively to the barbed ends of actin filaments; 2) the affinity of α1β heterodimers for actin (K_D = 1.6 × 10^-10 M) is ∼4-fold higher than that of α2β heterodimers (K_D = 6.3 × 10^-10 M); 3) the amino-terminal 40% of the α1 subunit (amino acids 1-115) and the ammo-terminal 31% of the β 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 a subunit appear to be required for binding to actin, as are the carboxyl-terminal 15 amino acids of the β subunit.