Regulation of Arabidopsis PI4P 5-kinases by phosphorylation

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Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) is a regulatory phospholipid that specifically controls localization or activity of target proteins with housekeeping functions or during stress-adaptation. PtdIns(4,5)P2 is formed by action of PI4P 5-kinases, which occur as a family of 11 isoenzymes in Arabidopsis. We have identified several PI4P 5- kinases with important functions in Arabidopsis. In various eukaryotic models PI4P 5-kinases are regulated by posttranslational modification and it has been shown that different Arabidopsis PI4P 5-kinases can be phosphorylated. However, so far it remains unclear i) what events might trigger phosphorylation, ii) what amino acids are phosphorylated, and iii) what consequences are regarding localization and functionality of the enzymes in the plants. Thus, the phosphorylation of PI4P 5-kinases in Arabidopsis plants will be investigated. The sites of phosphorylation will be determined and the respective amino acids changed to alanine or aspartate residues, constitutively mimicking the dephosphorylated or phosphorylated states, respectively. Recombinant variant proteins will be biochemically characterized in vitro. The subcellular localization of fluorescence-tagged proteins will be analyzed during transient expression in heterologous systems (Vicia faba guard cells and tobacco pollen tubes) and with stable expression in Arabidopsis. Physiological functionality will be tested by complementation of existing Arabidopsis T-DNA mutants.