Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and Chisholm, R. L. (1992) Cell 69, 951-962). Since normal myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the NH 2 -terminal 11 or 28 amino acid residues (⌬N11 or ⌬N28) or the COOH-terminal 15 amino acid residues (⌬C15) abolished binding of the ELC to the MHC when the mutants were expressed in wild-type (WT) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less efficient competition with WT ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE ؊ ) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, ⌬N28 and ⌬N11 bound to the MHC at 15% of WT levels and ⌬C15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE ؊ cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo.Myosin is a mechanochemical enzyme that generates force during muscle contraction and has a fundamental role in a variety of cell movements (for review, see Ref. 2). Conventional myosin is a hexameric protein composed of a pair of heavy chains (MHC) 1 and two pairs of light chains, called essential light chains (ELC) and regulatory light chains (RLC). Both light chains bind to the neck of myosin (3, 4). We have shown that ELC-deficient Dictyostelium cell lines are defective in cytokinesis, and myosin purified from these cells does not show significant actin-activated ATPase activity (1). Since normal myosin function requires association of the ELC, we set out to map the domains of the ELC that are critical for ELC-MHC interaction.Sequence comparisons of ELCs from various sources show that the COOH-terminal half of the molecule is more conserved than the NH 2 -terminal half (5, 6) and that it may represent the site required for association with MHC. Skeletal muscle alkali light chain A1 in which the COOH-terminal 14 residues were chemically removed did not bind the S1 heavy chain, although the conformation remained largely unchanged (7). Transfection studies also showed that a COOH-terminal truncation of the alkali light chain A1 failed to colocalize with acto-myosin structures in cultured myocytes (8).The ELC belongs to the calmodulin-troponin gene family, which contains four helix-loop-helix structures, also known as EF-hand...

We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7–11, in which the expression of ELC was inhibited by antisense RNA overexpression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting. The mlcE- mutants provide a clean genetic background for phenotypic analysis and biochemical characterization by removing complications arising from the residual ELC present in 7–11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome. The mlcE- mutants, when grown in suspension, exhibited the typical multinucleate phenotype observed in both myosin heavy chain mutants and 7–11 cells. This phenotype was rescued by introducing a construct that expressed the wild-type Dictyostelium ELC cDNA. Myosin purified from the mlcE- cells exhibited significant calcium ATPase activity, but the actin-activated ATPase activity was greatly reduced. The results obtained from the mlcE- mutants strengthen our previous conclusion based on the antisense cell line 7–11 that ELC is critical for myosin function. The proper localization of myosin in mlcE- cells suggests that its phenotypic defects primarily arise from defective contractile function of myosin rather than its mislocalization. The enzymatic defect of myosin in mlcE- cells also suggests a possible mechanism for the observed chemotactic defect of mlcE- cells. We have shown that while mlcE- cells were able to respond to chemoattractant with proper directionality, their rate of movement was reduced. During chemotaxis, proper directionality toward chemoattractant may depend primarily on proper localization of myosin, while efficient motility requires contractile function. In addition, we have analyzed the morphogenetic events during the development of mlcE- cells using lacZ reporter constructs expressed from cell type specific promoters. By analyzing the morphogenetic patterns of the two major cell types arising during Dictyostelium development, prespore and prestalk cells, we have shown that the localization of prespore cells is more susceptible to the loss of ELC than prestalk cells, although localization of both cell types is abnormal when developed in chimeras formed by mixing equal numbers of wild-type and mutant cells. These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin.