Nonspecific weak actomyosin interactions: Relocation of charged residues in subdomain 1 of actin does not alter actomyosin function

Yeast actin mutants with relocated charged residues within subdomain 1 were constructed so we could investigate the functional importance of individua l clusters of acidic residues in mediating actomyosin weak-binding states i n the cross-bridge cycle. Past studies have established a functional role f or three distinct pairs of charged residues within this region of yeast act in (D2/E4, D24/D25, and E990/E100); the loss of any one of these pairs resu lted in the same impairment in weak actomyosin interaction and in its funct ion. However, the specificity of myosin interaction with these sites has no t yet been addressed. To investigate this, we made and analyzed two new act in mutants, 4Ac/D24A/D25A and 4Ac/E99A/E100A. In these mutants, the acidic residues of the D24/D25 or E99/E100 sites were replaced with uncharged resi dues (alanines) and a pair of acidic residues was inserted at the N-terminu s, maintaining the overall charge density of subdomain 1. Using the in vitr o motility assays, we found that the sliding and force generation propertie s of these mutant actins were identical to those of wild-type actin. Simila rly, actin-activated ATPase activities of the mutant and wild-type actins w ere also indistinguishable. Additionally, the binding of S1 to these mutant actins in the presence of ATP was similar to that of wild-type actin. Thes e results show that relocation of charged residues in subdomain 1 of actin does not affect the weak actomyosin interactions and actomyosin function.