A hydrophilic cation-binding protein of Arabidopsis thaliana, AtPCaP1, is localized to plasma membrane via N-myristoylation and interacts with calmodulin and the phosphatidylinositol phosphates PtdIns(3,4,5) P3 and PtdIns(3,5) P2.

A hydrophilic cation-binding protein, PCaP1, was found to be stably bound to the plasma membrane in Arabidopsis thaliana. PCaP1 was quantified to account for 0.03–0.08% of the crude membrane fractions from roots and shoots. Its homologous protein was detected in several plant species. We investigated the mechanism of membrane association of PCaP1 by transient expression of fusion protein with green fluorescent protein. The amino-terminal sequence of 27 residues of PCaP1 had a potential to localize the fusion protein with green fluorescent protein to the plasma membrane, and the substitution of Gly at position 2 with Ala resulted in the cytoplasmic localization of PCaP1. When PCaP1 was expressed in the in vitro transcription/translation system with [3H]myristic acid, the label was incorporated into PCaP1, but not into a mutant PCaP1 with Gly2 replaced by Ala. These results indicate that PCaP1 tightly binds to the plasma membrane via N-myristoylation at Gly2. We examined the binding capacity with phosphatidylinositol phosphates (PtdIns Ps), and found that PCaP1 selectively interacts with phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate. Competition assay with the N-terminal peptide and mutational analysis revealed that PCaP1 interacts with these two PtdIns Ps at the N-terminal part. Interaction of PCaP1 with the membrane and PtdIns Ps was not altered in the presence of Ca2+ at physiological concentrations. Furthermore, calmodulin associated with PCaP1 in a Ca2+-dependent manner, and its association weakened the interaction of PCaP1 with PtdIns Ps. These results indicate that the N-terminal part is essential for both N-myristoylation and interaction with PtdIns Ps, and that PCaP1 may be involved in intracellular signalling through interaction with PtdIns Ps and calmodulin. [ABSTRACT FROM AUTHOR]