SUBSTITUTION MUTATIONS IN THE MYOSIN ESSENTIAL LIGHT-CHAIN LEAD TO REDUCED ACTIN-ACTIVATED ATPASE ACTIVITY DESPITE STOICHIOMETRIC BINDING TO THE HEAVY-CHAIN

Myosin essential light chain (ELC) wraps around an alpha-helix that ex tends from the myosin head, where it is believed to play a structural support role. To identify other role(s) of the ELC in myosin function, we have used an alanine scanning mutagenesis approach to convert char ged residues in loops I, II, III, and helix G of the Dictyostelium ELC into uncharged alanines, Dictyostelium was used as a host system to s tudy the phenotypic and biochemical consequences associated with the m utations, The ELC carrying loop mutations bound with normal stoichiome try to the myosin heavy chain when expressed in ELC-minus cells. When expressed in wild type cells these mutants competed efficiently with t he endogenous ELC for binding, suggesting that the affinity of their i nteraction with the heavy chain is comparable to that of wild type, Ho wever, despite apparently normal association of ELC the cells still ex hibited a reduced efficiency to undergo cytokinesis in suspension. Myo sin purified from these cells exhibited 4-5-fold reduction in actin-ac tivated ATPase activity and a decrease in motor function as assessed b y an in vitro motility assay, These results suggest that the ELC contr ibutes to myosin's enzymatic activity in addition to providing structu ral support for the Lu-helical neck region of myosin heavy chain.