During chemotactic stimulation of amoebae of Dictyostelium discoideum two proteins are selectively incorporated into the actin cytoskeleton. ABP-120 has been implicated in pseudopod extension by in vitro activity, immunolocalization and mutant analysis. ABP-50 has been implicated in filopod extension by in vitro activity and immunolocalization. ABP-50 is also Dictyostelium EF-1alpha suggesting a relationship between the actin cytoskeleton and protein synthesis. The amino terminal sequence of ABP-120 is highly conserved in many actin cross-linking proteins. We will map the dimerization domain of ABP-120 and determine the relative contribution of actin binding and filament cross- linking to the function of ABP-120 in generating part of the force of pseudopod extension. These studies will be done by expression of truncated ABP-120 in existing ABP-120- mutants. In related studies we will clone and sequence ABP-240, a protein with a similar structure to that of ABP-120, and prepare ABP-240- mutants and ABP-120-/ABP-240- double mutants whose phenotypes will be compared to that of ABP-120- mutants. Preliminary evidence indicates that the protein synthetic activity of ABP- 50 is regulated by actin in such a way that protein synthesis may occur predominantly in association with F-actin in the cytoskeleton. To understand the mechanisms of regulation of protein synthetic activity of ABP-50 by actin and its cytoskeletal association following chemotactic stimulation, we will map the actin binding sites of ABP-50 by protein chemical and molecular genetic techniques, determine the mechanism of inhibition of the protein synthetic activity of ABP-50 by G-actin, and determine the effect of mutations of the actin binding sites on the protein synthetic activity of ABP-50.
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