Genetically engineered truncated myosin in Dictyostelium: the carboxyl-terminal regulatory domain is not required for the developmental cycle.
The study of engineered Dictyostelium mutants with altered or missing myosin has revealed the molecule to be essential both for cytokinesis and for completion of the complex Dictyostelium developmental cycle. To explore the biological role of the carboxyl-terminal portion of the myosin tail, we have created a Dictyostelium cell line bearing a mutation designated my delta C34 in the myosin (mhcA) locus. This cell line produces a truncated myosin protein lacking the 34-kDa carboxyl terminus of the wild-type tail. Southern blots of the mutant cells show that the myosin gene was disrupted by homologous recombination of the transforming plasmid into the myosin locus. Based on in vitro studies of myosin functional domains, the 200-kDa truncated myosin was designed to include a domain important for assembly but to eliminate a domain important for threonine phosphorylation. The mutant cells are defective in cytokinesis, similar to those mutants that are either devoid of myosin (null cells) or contain a truncated 140-kDa myosin (hmm cells). However, unlike previous mutants, the cells carrying the my delta C34 mutation are able to complete the Dictyostelium developmental cycle to form fruiting bodies. Thus a truncated 200-kDa myosin can substitute for native myosin to function in developing cells. These results demonstrate that the 34-kDa carboxyl terminus of myosin, which contributes regulated phosphorylation sites and 20% of the total length of the rod, is not required for the developmental cycle of Dictyostelium.
O'Halloran
T J
TJ
Department of Cell Biology, Stanford University School of Medicine, CA 94305.
Spudich
J A
JA
D015139:Blotting, Southern; D002453:Cell Cycle; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000254:growth & development*; D005818:Genetic Engineering; D009154:Mutation; D009218:Myosins / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping
D004271:DNA, Fungal; D009218:Myosins