Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation.
The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.
Department of Cell Biology, Stanford University School of Medicine, California 94305.
T J
TJ
O'Halloran
S
S
Ravid
J A
JA
Spudich
D003001:Cloning, Molecular; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008854:Microscopy, Electron; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D009218:Myosins / Q000235:genetics* / Q000648:ultrastructure; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000648:ultrastructure
D015879:Myosin Subfragments; D011994:Recombinant Proteins; D009218:Myosins