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.

Plant microtubules (MTs) play essential roles in cell division, anisotropic cell expansion, and overall organ morphology. Microtubule-associated proteins (MAPs) bind to MTs and regulate their dynamics, stability, and organization. Identifying the full set of MAPs in plants would greatly enhance our understanding of how diverse MT arrays are formed and function; however, few proteomics studies have characterized plant MAPs. Using liquid chromatography-tandem mass spectrometry, we identified hundreds of proteins from MAP-enriched preparations derived from cell suspension cultures of Arabidopsis (Arabidopsis thaliana). Previously reported MAPs, MT regulators, kinesins, dynamins, peroxisome-resident enzymes, and proteins implicated in replication, transcription, and translation were highly enriched. Dozens of proteins of unknown function were identified, among which 12 were tagged with green fluorescent protein (GFP) and examined for their ability to colocalize with MTs when transiently expressed in plant cells. Six proteins did indeed colocalize with cortical MTs in planta. We further characterized one of these MAPs, designated as BASIC PROLINE-RICH PROTEIN1 (BPP1), which belongs to a sevenmember family in Arabidopsis. BPP1-GFP decorated interphase and mitotic MT arrays in transgenic Arabidopsis plants. A highly basic, conserved region was responsible for the in vivo MT association. Overexpression of BPP1-GFP stabilized MTs, caused right-handed helical growth in rapidly elongating tissues, promoted the formation of transverse MT arrays, and resulted in the outgrowth of epidermal cells in light-grown hypocotyls. Our high-quality proteome database of Arabidopsis MAP-enriched preparations is a useful resource for identifying novel MT regulators and evaluating potential MT associations of proteins known to have other cellular functions.Microtubules (MTs) are major structural components of the plant cytoskeleton that are composed of a-tubulin/b-tubulin heterodimer subunits and are intricately involved in cell division, morphology, and intracellular organization and transport. The ability of the MT cytoskeleton to fulfill its versatile cellular functions relies on its intrinsically dynamic polymer properties. Individual MTs alternate between phases of growth and shrinkage by rapidly attaching and removing tubulin subunits at their ends. These cycles of polymerization and depolymerization, which continuously reorganize the MT cytoskeleton, are sometimes interrupted by pauses in which neither growth nor shrinkage occurs (Desai and Mitchison,

Addendum to: Kato M, Nagasaki-Takeuchi N, Ide Y, Maeshima M. An Arabidopsis hydrophilic Ca 2+ -binding protein with a PEVK-rich domain, PCaP2, is associated with the plasma membrane and interacts with calmodulin and phosphatidylinositol phosphates.

PCaP1, a hydrophilic cation-binding protein, is bound to the plasma membrane in Arabidopsis thaliana. We focused on the physicochemical properties of PCaP1 to understand its uniqueness in terms of structure and binding of metal ions. On fluorescence analysis, PCaP1 showed a signal of structural change in the presence of Cu(2+). The near-UV CD spectra showed a marked change of PCaP1 in CuCl(2) solution. The far-UV CD spectra showed the presence of alpha-helices and the intrinsically unstructured region. However, addition of Cu(2+) gave no change in the far-UV CD spectra. These results indicate that Cu(2+) induced a change in the tertiary structure without changing the secondary structure. The protein was sensitive to proteinase in the presence of Cu(2+), supporting that Cu(2+) is involved in the structural change. The PCaP1 solution was titrated with CuCl(2) and the change in the fluorescence spectrum was monitored to characterize Cu(2+)-binding properties. The obtained values of K(d) for Cu(2+) and the ligand-binding number were 10 microM and six ions per molecule, respectively. These findings indicate that PCaP1 has a high Cu(2+)-binding capacity with a relatively high affinity. PCaP1 lacks cysteine and histidine residues. A large number of glutamate residues may be involved in the Cu(2+) binding.

IntroducitionRadioisotopes (RI) such as 3 H, 14 C, 32 P, and 45 Ca are excellent tools in biological research. Most RI are used as tracers in studies of primary and secondary metabolism, drug metabolism, transcription, translation, post-translational modifications such as protein phosphorylation, association of proteins with metals, and transport of metals across biomembranes. Furthermore, some experiments have used neutrons for mutagenesis of microorganisms, animals, and plants. Recent progress in the biological sciences has resulted in novel probes and labeling reagents, which has decreased the need for RI. Experiments with RI require experimental space specialized for RI, careful experimental procedures, and training. Although these are disadvantages, RI are still useful and powerful tools with high resolution compared with non-RI methods. Here, we describe the advantages of RI in biochemical assays, and detailed experimental procedures of metal-binding assays and membrane transport measurements of metal cations, especially calcium and zinc. Advantages of radioisotopes as tracersMost metabolic pathways that are described in biochemistry textbooks, in various organisms including humans, plants, and microorganisms, could not have been determined without RI such as 14 C, 35 S, 32 P, and 3 H. Biochemical experiments with RI provide information on the fates of metabolites, nutrients, and inorganic ions at each periodic stage of living organisms or cells. In the early era of molecular biology, 32 P was used as an essential tool in a large number of laboratories to determine DNA sequences and to identify target DNAs or mRNAs. Phosphorylation of serine and/or tyrosine residue s i s a key covalent modification of proteins. 32 P ([-32 P]ATP) is still used to investigate this biochemical process. 35 Antibodies specific to phosphoserine, phosphotyrosine, or peptides containing phosphorylated amino acid residues are prepared and used. The accuracy and sensitivity depend on the quality and specificity of the antibodies. In most cases, researchers must pay attention to artifactual signals. In contrast, labeling proteins with RI provides clear and quantitative information on protein phosphorylation. In RI methods, proteins are phosphorylated with [-32 P]ATP in cell free systems (in vitro) or in experiments using cells, tissues, or organisms (in vivo), and then proteins are extracted and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins in the gel are transferred onto a transfer membrane such as polyvinylidine fluoride (PVDF). The membrane is dried and subjected to autoradiography by contact with an X-ray film at 80C for a few days. Imaging plates are now generally used for the detection of phosphorylated proteins. Imaging plates have several advantages compared with autoradiography: (i) high sensitivity (quick detection), (ii) good linearity between the content of 32 P and the signal, (iii) digital imaging, and (iv) no requirement of a dark room. 3. Reflection of natural con...