Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment.
The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.
De Lozanne
A
A
Department of Cell Biology, Stanford University School of Medicine, California 94305.
Berlot
C H
CH
Leinwand
L A
LA
Spudich
J A
JA
D004023:Dictyostelium / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000235:genetics; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000235:genetics*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism
D015879:Myosin Subfragments; D010446:Peptide Fragments; D010769:Phosphothreonine; D009219:Myosin-Light-Chain Kinase; D009218:Myosins