Regulatory light chain mutations affect myosin motor function and kinetics

The actin-based motor protein myosin II plays a critical role in many cellu lar processes in both muscle and nonmuscle cells. Targeted disruption of th e Dictyostelium regulatory light chain (RLC) caused defects in cytokinesis and multicellular morphogenesis. In contrast, a myosin heavy chain mutant l acking the RLC binding site, and therefore bound RLC, showed normal cytokin esis and development. One interpretation of these apparently contradictory results is that the phenotypic defects in the RLC null mutant results from mislocalization of myosin caused by aggregation of RLC null myosin. To dist inguish this from the alternative explanation that the RLC can directly inf luence myosin activity, we expressed three RLC point mutations (E12T, G18K and N94A) in a Dictyostelium RLC null mutant. The position of these mutatio ns corresponds to the position of mutations that have been shown to result in familial hypertrophic cardiomyopathy in humans. Analysis of purified Dic tyostelium myosin showed that while these mutations did not affect binding of the RLC to the MHC, its phosphorylation by myosin light chain kinase or regulation of its activity by phosphorylation, they resulted in decreased m yosin function. All three mutants showed impaired cytokinesis in suspension , and one produced defective fruiting bodies with short stalks and decrease d spore formation. The abnormal myosin localization seen in the RLC null mu tant was restored to wild-type localization by expression of all three RLC mutants. Although two of the mutant myosins had wild-type actin-activated A TPase, they produced in vitro motility rates half that of wild type. N94A m yosin showed a fivefold decrease in actin-ATPase and a similar decrease in the rate at which it moved actin in vitro. These results indicate that the RLC can play a direct role in determining the force transmission and kineti c properties of myosin.