Both the amino and carboxyl termini of Dictyostelium myosin essential light 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 (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 NH2-terminal 11 or 28 amino acid residues (delta N11 or delta N28) or the COOH-terminal 15 amino acid residues (delta 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, delta N28 and delta N11 bound to the MHC at 15% of WT levels and delta 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.
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003599:Cytoskeleton / Q000648:ultrastructure; D004251:DNA Transposable Elements; D004023:Dictyostelium / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
D004251:DNA Transposable Elements; D018994:Myosin Light Chains; D011994:Recombinant Proteins; D018995:Myosin Heavy Chains