PURIFICATION AND CHARACTERIZATION OF A PHOSPHATIDYLINOSITOL 4-KINASE ACTIVATOR IN CARROT CELLS

A phosphatidylinositol 4-kinase activator (PIK-A49) has been purified from carrot cells grown in suspension culture. The activator was purified from a soluble fraction using DEAE-Sepharose CL-6B and S-Sepharose chromatography columns. PIK-A49 has a relative molecular mass of 49 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A50 for the activation of the Triton X-100-solubilized phosphatidylinositol 4-kinase fraction was 0.1 muM. Maximal activation was 3-4-fold. The analysis of the sequences of seven peptide fragments containing a total of 142 amino acid residues indicated that PIK-A49 was 69% identical to an actin-binding protein (ABP-50) from Dictyostelium and >90% identical to elongation factor-1alpha (EF-1alpha) from carrot, tomato, and Arabidopsis. PIK-A49 bound actin and facilitated actin polymerization. Poly(U)-directed polyphenylalanine synthesis assays indicated that PIK-A49 had EF-1alpha activity. The EF-1alpha activity was enhanced by rabbit EF-1betagamma. Activation of phosphatidylinositol 4-kinase by a protein that binds actin and that has EF-1alpha activity provides additional complexity to the signal transduction mechanisms involving inositol phospholipid metabolism.