BOTH THE AMINO AND CARBOXYL TERMINI OF DICTYOSTELIUM MYOSIN ESSENTIALLIGHT-CHAIN ARE REQUIRED FOR BINDING TO MYOSIN HEAVY-CHAIN

Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollent, R. S., Che n, T. L., Trivinos-Lagos, L., and Chisholm, R. L. (1992) Cell 69, 951- 962). Since normal myosin function requires association of ELC, we inv estigated the domains of ELC that are necessary for binding to the myo sin heavy chain (MHC), Deleting the NH2-terminal 11 or 28 amino acid r esidues (Delta N11 or Delta N28) or the COOH terminal 15 amino acid re sidues (Delta C15) abolished binding of the ELC to the MHC when the mu tants were expressed in wild-type (WT) cells, In contrast, the ELC car rying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less ef ficient competition with WT ELC suggested that the affinity for the MH C is reduced, When these mutants were ex pressed in ELC-minus (mlcE(-) ) cells, where the binding to the heavy chain is not dependent on effi cient competition with the endogenous ELC, Delta N28 and Delta N11 bou nd to the MHC at 15% of WT levels and Delta C15 did not bind to a sign ificant degree, 14 and D4, however, bound with normal stoichiometry. T hese data indicate that residues at both termini of the ELC are requir ed for association with the MHC, while the central linker domain appea rs 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 obs erved, indicating that a stoichiometric ELC MHC association is necessa ry for normal myosin function in vivo.