MUTATIONAL ANALYSIS OF THE ROLE OF THE N-TERMINUS OF ACTIN IN ACTOMYOSIN INTERACTIONS - COMPARISON WITH OTHER MUTANT ACTINS AND IMPLICATIONS FOR THE CROSS-BRIDGE CYCLE

Yeast actin mutants with acidic residues at the N terminus either neut ralized (DNEQ) or deleted (Delta-DSE) were used to assess the role of N-terminal acidic residues in the interactions of actin with myosin in the contractile cycle. Cosedimentation experiments revealed an simila r to 3-fold decrease in the binding constant for DNEQ and Delta-DSE ac tins to myosin subfragment-1 (S1) relative to that of wild type actin both in the presence of MgATP and in the absence of nucleotides (stron g binding). DNEQ and Delta-DSE actins protected S1 from tryptic digest ion as well as the wild type and rabbit actins. The activation of S1 A TPase by DNEQ and Delta-DSE actins (up to 50 mu M) was very low but in creased greatly after cross-linking these mutant actins to S1 by dimet hyl suberimidate. Thus, the increased dissociation of mutant actins fr om S1 in the presence of ATP is the main cause for the low acto-S1 ATP ase activities. At low-ionic strength conditions and in the presence o f methylcellulose, the DNEQ and Delta-DSE actins moved in the in vitro motility assays at a mean velocity similar to that of wild type actin (3.0 mu m/s). Yet, the sliding velocity of the N-terminal and D24A/D2 5A and E99A/E100A mutant actins decreased relative to that of the wild type at all levels of external load introduced into the assay and at low densities of heavy meromyosin (HMM) on the cover slip. This indica tes a lower relative force generation with the mutant actins. In contr ast, the force generated under the same conditions with the 4Ac mutant actin (with four acidic charges at the N terminus) was higher than wi th wild type actin. At higher-ionic strength conditions (I = 150 mM), the sliding of the DNEQ and Delta-DSE as well as that of the D24A/D25A and E99A/E100A actins ceased even in the presence of methylcellulose, while I341A actin (deficient in strong binding to myosin) still moved . These results indicate the importance of electrostatic actomyosin in teractions under physiological salt conditions and show functionally d istinct roles for the different myosin binding sites on actin.