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

Abstract

The intracellular localization of proteins is critical for expression of their cellular function, and is determined by several mechanisms, including their primary sequences, post-translational processing, covalent modifications and affinity to other elements. Most soluble proteins are localized to the cytoplasm, intra-organelle spaces, cytoskeletons or secreted out of the cells. However, some parts of hydrophilic proteins in cells can be 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 [ 3 H]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 (PtdInsPs), 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 PtdInsPs at the N-terminal part. Interaction of PCaP1 with the membrane and PtdInsPs was not altered in the presence of Ca 2+ at physiological concentrations. Furthermore, calmodulin associated with PCaP1 in a Ca 2+ -dependent manner, and its association weakened the interaction of PCaP1 with PtdInsPs. These results indicate that the N-terminal part is essential for both N-myristoylation and interaction with PtdInsPs, and that PCaP1 may be involved in intracellular signalling through interaction with PtdInsPs and calmodulin.Abbreviations CaM, calmodulin; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; MAP, methionine aminopeptidase; NMT, myristoylCoA:protein N-myristoyltransferase; PCaP1, plasma membrane-associated cation-binding protein; PtdIns(3,4,5)P 3 , phosphatidylinositol 3,4,5-triphosphate; PtdIns(3,5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdInsP, phosphatidylinositol phosphate.

MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules inArabidopsis

“…Although MDP25 is a protein in Arabidopsis that is homologous with MAP18 (Nagasaki et al, 2008;Kato et al, 2010a), there is no evidence that it directly affects microtubules. To determine whether this protein binds to microtubules, we expressed MDP25 in bacterial cells and purified the fusion protein.…”

“…It is reported that two hydrophilic cation binding proteins, Arabidopsis plasma membrane-associated cation binding protein 1 (PCaP1) and PCaP2, stably bind to the plasma membrane and to calcium (Ide et al, 2007;Nagasaki et al, 2008;Kato et al, 2010a). PCaP2 is a microtubule-associated protein previously named MAP18, which binds and regulates microtubules (Wang et al, 2007).…”

“…PCaP2 is a microtubule-associated protein previously named MAP18, which binds and regulates microtubules (Wang et al, 2007). PCaP1 is homologous with MAP18 in Arabidopsis and exhibits similar biochemical properties as MAP18 with regard to cation binding in vitro (Nagasaki et al, 2008;Kato et al, 2010a). However, it is unknown whether PCaP1 is a novel microtubule regulatory protein and how it functions in plant cells.…”

Microtubules in Plants

“…The Arabidopsis plasma membrane-associated Ca 2+ -binding proteins PCaP1 (At4g20260) and PCaP2 (At5g44610) bind to the Ca 2+ -calmodulin complex and phosphatidylinositol phosphates (Nagasaki et al 2008;Kato et al, 2010). Interestingly, recombinant PCaP proteins depolymerize, in a Ca 2+ -dependent manner, both MTs (thus called MAP18 and MT-Destabilizing Protein 25; Li et al 2011) and actin filaments (Zhu et al 2013;Qin et al 2014).…”

Requirement of Calcium Binding, Myristoylation, and Protein-Protein Interaction for the Copine BON1 Function in Arabidopsis

“…Myristoylation is found to target some plant proteins to membranes. For instance, a hydrophilic cation-binding protein AtPCaP1, and a calcineurin B-like (CBL1) could localize to the plasma membrane through this lipid modification (15,19,20). The role of this potential modification in BON1 is unknown.…”