An Arabidopsis Hydrophilic Ca2+-Binding Protein with a PEVK-Rich Domain, PCaP2, is Associated with the Plasma Membrane and Interacts with Calmodulin and Phosphatidylinositol Phosphates

Abstract

We found a new hydrophilic protein in Arabidopsis thaliana. Real-time PCR demonstrated that the protein was expressed in roots. Histochemical analysis of promoter-beta-glucuronidase fusions demonstrated its extensive expression in root hairs. The protein is rich in proline, glutamate, valine and lysine residues (PEVK-rich domain), and bound Ca(2+) even in the presence of Mg(2+) and K(+) when examined by the (45)Ca overlay assay. Treatment of seedlings with K(+), Mn(2+), Zn(2+), Na(+), ABA and gibberellic acid, and cold and drought stresses enhanced the transcription. Expression of the protein linked to green fluorescent protein in A. thaliana showed its plasma membrane localization and cell-specific expression in the epidermal cells including root hairs and the elongating pollen tubes. Therefore, we named the protein PCaP2 (plasma membrane-associated Ca(2+)-binding protein-2). The substitution of glycine at position 2 with alanine resulted in cytoplasmic localization of PCaP2. These results and the N-terminal characteristic motif suggest that PCaP2 is N-myristoylated at Gly2. We examined the capacity for binding to phosphatidylinositol phosphates (PtdInsPs), and found that PCaP2 interacts strongly with PtdIns(3,5)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), and weakly with PtdIns(3,4)P(2). Furthermore, calmodulin was associated with PCaP2 in a Ca(2+)-dependent manner, and its association weakened the interaction of PCaP2 with PtdInsPs. These results indicate that PCaP2 is involved in intracellular signaling through interaction with PtdInsPs and calmodulin in growing root hairs. PCaP2 was previously reported as microtubule-associated protein-18. We discuss the physiological roles of PCaP2 in relation to microtubules in cells.

The conserved mobility of mitochondria during leaf senescence reflects differential regulation of the cytoskeletal components inArabidopsis thaliana

“…5 Interestingly, a strong induction of MAP18 was similarly observed in individually-darkened leaves and in leaves from entirely darkened plants. However, while a disruption of the MT network was observed in the epidermis and mesophyll cells from leaves undergoing dark-induced senescence, the MT network was rather well conserved in leaves from entirely darkened plant.…”

“…However, while a disruption of the MT network was observed in the epidermis and mesophyll cells from leaves undergoing dark-induced senescence, the MT network was rather well conserved in leaves from entirely darkened plant. 1 Therefore, although MAP18/ PCaP2 (as renamed by Kato et al 5 ) is very likely to be involved in the regulation of cellular functions including stress response and cytoskeletal reorganization, Figure 1. representative mature rosette leaves undergoing natural senescence.…”

Leaf Senescence Is Accompanied by an Early Disruption of the Microtubule Network in Arabidopsis

“…However, our analysis showed that MAP18 was also induced in leaves from whole darkened plants, whereas only a minor destabilization of the MT network was observed. Furthermore, in a recent study, Kato et al (2010) investigated the biochemical properties of MAP18 and concluded that this protein was a plasma membrane-associated Ca 2+ -binding protein (consequently renamed PCaP2 by the authors) and was very likely to be involved in the regulation of cellular functions, including stress response and cytoskeletal reorganization. In vivo, MAP18/PCaP2 appeared to be associated with the plasma membrane due to its N-myristoylation and strongly interacted with several phosphatidylinositol phosphates in a Ca 2+ -calmodulindependent manner (Kato et al, 2010).…”

“…Furthermore, in a recent study, Kato et al (2010) investigated the biochemical properties of MAP18 and concluded that this protein was a plasma membrane-associated Ca 2+ -binding protein (consequently renamed PCaP2 by the authors) and was very likely to be involved in the regulation of cellular functions, including stress response and cytoskeletal reorganization. In vivo, MAP18/PCaP2 appeared to be associated with the plasma membrane due to its N-myristoylation and strongly interacted with several phosphatidylinositol phosphates in a Ca 2+ -calmodulindependent manner (Kato et al, 2010). We consequently conclude that increased expression of MAP18 is not sufficient to destabilize the MT network in vivo but could instead be part of a more complex Ca 2+ -dependent signaling mechanism potentially leading to the perturbation of the MT cytoskeleton.…”

Application of Radioisotopes in Biochemical Analyses: Metal Binding Proteins and Metal Transporters

“…Radioisotope 45 Ca is necessary to confirm that the protein(s) of interest can bind calcium. The 45 Ca overlay assay is one convenient method (Campbell et al, 1983;Yuasa & Maeshima, 2002;Ide et al, 2007;Kato et al, 2010). If a purified preparation is available, aliquots of the purified sample are spotted onto a membrane filter such as a PVDF membrane (ca.…”