A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure

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.     

细胞膜相联钙结合蛋白的结构和功能研究进展

细胞膜相联钙结合蛋白(PCaP)是定位于细胞膜上的一类新的Ca2+结合蛋白。简要综述了PCaP的蛋白结构、调控Ca2+/磷脂酰肌醇3-磷酸信号通路的分子机制、调节微管微丝稳定的能力,以及其在植物顶端生长极性中的功能研究进展。     

An endogenous inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase

An inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was purified to apparent homogeneity from rat cerebrum by a molecular weight cut followed by chromatography of cytosol proteins with molecular weights between 10 000 and 3500 on DEAE-Sephadex at pH 5.2. The inhibitor could be partially inactivated by proteinases and dithiothreitol, but was heat-stable. Gel filtration gave a molecular weight of about 6000. Like the (Ca2+ + Mg2+)-ATPase inhibitor protein isolated from erythrocytes, the inhibitor from brain contains a characteristic high proportion of glutamic acid (36%) and glycine (37%) residues. Synaptic plasma membrane Mg2+-ATPase and microsomal membrane (Ca2+ + Mg2+)-ATPase did not respond to the inhibitor. Synaptic plasma membrane and erythrocyte membrane (Ca2+ + Mg2+)-ATPases, however, were affected. Inhibitory influence on synaptic membrane (Ca2+ + Mg2+)-ATPase was reversible, since inhibition could be relieved upon removal of inhibitor from saturable sites on the membrane. The inhibitor is not a calmodulin-binding protein, since the concentration of calmodulin for half-maximal activation of the ATPase was unaffected by its presence. Mode of inhibition of the (Ca2+ + Mg2+)-ATPase by the inhibitor was non-competitive.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg2+)-ATPase activity in islets of Langerhans

Studies were undertaken to determine whether factors which affect insulin secretion may exert their effects by altering the activity of an islet-cell plasma membrane Ca2+ extrusion pump. The insulin secretagogue, D-glucose, and a variety of phosphorylated hexoses, glucose 6-P, glucose 1,6-P, fructose 6-P, and fructose 2,6-P, were evaluated for their effect on an islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase and were found to be ineffective in altering enzyme activity. D-Glucose also did not alter the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Similarly, cAMP, the catalytic subunit of cAMP-dependent protein kinase, arachidonic acid, or prostaglandin E2 did not affect either the plasma membrane (Ca2+ + Mg2+)-ATPase or the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Whereas previous studies have suggested that D-glucose and/or cAMP may inhibit ATPase activities in islets, these results indicate that the agents, i.e., D-glucose and cAMP, which stimulate and/or potentiate insulin secretion from the islet cell, do not modify Ca2+ fluxes by directly regulating the islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase. In contrast, the acidic phospholipids, phosphatidic acid and phosphatidylserine, stimulated the enzyme activity in a concentration-dependent manner whereas phosphatidylcholine had only a minimal effect. The diacylglycerol, dilinolein, stimulated the (Ca2+ + Mg2+)-ATPase activity in the presence of phosphatidylserine, but not in the absence of phospholipids. These effects were independent of phospholipid-stimulated protein phosphorylation in the islet-cell plasma membrane under the conditions of the ATPase assay.     

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.     

The animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases--approaches to molecular arrangements of functional parts and oxidative changes.

The molecular structures of animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases are not completely understood in part due to the fact that no suitable single crystal is available. The elucidation of the two-dimensional structure is in progress. The amino acid sequences of human erythrocyte and rat plasma membrane Ca2+ pump isoforms as well as of the pig smooth muscle plasma membrane Ca2+ pump are already known. This article reviews the present state of the knowledge in (Ca(2+)+Mg2+)-ATPase research of animal and human plasma membranes performed in the past few years, concerning in particular arrangements of proteolytically cleaved fragments, and relations between the erythrocyte (Ca(2+)+Mg2+)-ATPase in situ and the purified red cell enzyme, oxidative changes. Results of different experimental approaches concerning the structure of (Ca(2+)+Mg2+)-ATPases rather than the applications of the methods used are emphasized.     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Utilization of purified myometrium cell plasma membrane Ca2+, Mg2+-ATPase for comparative estimation of the efficiency of energy-dependent Ca2+-transport inhibitors

With the aim of comparative estimation of efficacy of well-known inhibitors of energy-dependent Ca(2+)-transporting systems their effects were investigated on the activity of purified Ca2+, Mg(2+)-ATPase of the myometrium cell plasma membranes. From the approved inhibitors (eosin Y, o-vanadate, thapsigargin, cyclopiazonic acid, ruthenium red, sodium azide) only eosin Y and o-vanadate are potent inhibitors of myometrium sarcolemma Ca(2+)-pump: the values of Ki equal 0.8 and 4.7 microM, respectively. Thapsigargin and cyclopiazonic acid as well as ruthenium red in concentrations inhibiting, respectively, endo(sarco)plasmic reticulum Ca(2+)-pump and energy-dependent Ca(2+)-transport in mitochondria had no effect on the Ca2+, Mg(2+)-ATPase of the uterus smooth muscle cell plasma membrane. Sodium azide (10 mM) blocking completely Ca(2+)-transport in mitochondria inhibited activity of the plasma membrane Ca(2+)-transporting ATPase by 14%.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. II. Dependence on calcium and a cytoplasmic activator.

1. Activity of the (Ca2+ + Mg2+)-ATPase of erythrocyte membrane may be enhanced by a cytoplasmic protein activator. The presence of Ca2+ is necessary for the ionic strength-dependent interaction between the erythrocyte membrane and the activator. This is true no matter the purity of activator (unfractionated hemolysis supernatant or partially purified activator) or the major source of ionic strength (imidazole or NaCl). 2. When the endogenous activator enhances (Ca2+ + Mg2+)-ATPase activity of the erythrocyte membrane, there is a physical association between activator and membrane. This association is not disrupted by a decrease in ionic strength to 0.005 but is reversed by exposure to 5 mM ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 3. Activator binding necessary for enhancement of (Ca2+ + Mg2+)-ATPase activity may occur during preparation of membranes or during incubation for assay of ATPase.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Cytoplasmic calcium increases in response to changes in the gravity vector in hypocotyls and petioles of Arabidopsis seedlings

Plants respond to a large variety of environmental signals, including changes in the gravity vector (gravistimulation). In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation is known to increase the cytoplasmic free calcium concentration ([Ca(2+)](c)). However, organs responsible for the [Ca(2+)](c) increase and the underlying cellular/molecular mechanisms remain to be solved. In this study, using Arabidopsis seedlings expressing apoaequorin, a Ca(2+)-sensitive luminescent protein in combination with an ultrasensitive photon counting camera, we clarified the organs where [Ca(2+)](c) increases in response to gravistimulation and characterized the physiological and pharmacological properties of the [Ca(2+)](c) increase. When the seedlings were gravistimulated by turning 180 degrees, they showed a transient biphasic [Ca(2+)](c) increase in their hypocotyls and petioles. The second peak of the [Ca(2+)](c) increase depended on the angle but not the speed of rotation, whereas the initial peak showed diametrically opposite characters. This suggests that the second [Ca(2+)](c) increase is specific for changes in the gravity vector. The potential mechanosensitive Ca(2+)-permeable channel (MSCC) inhibitors Gd(3+) and La(3+), the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and the endomembrane Ca(2+)-permeable channel inhibitor ruthenium red suppressed the second [Ca(2+)](c) increase, suggesting that it arises from Ca(2+) influx via putative MSCCs in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Moreover, the second [Ca(2+)](c) increase was attenuated by actin-disrupting drugs cytochalasin B and latrunculin B but not by microtubule-disrupting drugs oryzalin and nocodazole, implying that actin filaments are partially involved in the hypothetical activation of Ca(2+)-permeable channels. These results suggest that the second [Ca(2+)](c) increase via MSCCs is a gravity response in the hypocotyl and petiole of Arabidopsis seedlings.     

A Novel Family of Magnesium Transport Genes in Arabidopsis

Magnesium (Mg(2+)) is the most abundant divalent cation in plant cells and plays a critical role in many physiological processes. We describe the identification of a 10-member Arabidopsis gene family (AtMGT) encoding putative Mg(2+) transport proteins. Most members of the AtMGT family are expressed in a range of Arabidopsis tissues. One member of this family, AtMGT1, functionally complemented a bacterial mutant lacking Mg(2+) transport capability. A second member, AtMGT10, complemented a yeast mutant defective in Mg(2+) uptake and increased the cellular Mg(2+) content of starved cells threefold during a 60-min uptake period. (63)Ni tracer studies in bacteria showed that AtMGT1 has highest affinity for Mg(2+) but may also be capable of transporting several other divalent cations, including Ni(2+), Co(2+), Fe(2+), Mn(2+), and Cu(2+). However, the concentrations required for transport of these other cations are beyond normal physiological ranges. Both AtMGT1 and AtMGT10 are highly sensitive to Al(3+) inhibition, providing potential molecular targets for Al(3+) toxicity in plants. Using green fluorescence protein as a reporter, we localized AtMGT1 protein to the plasma membrane in Arabidopsis plants. We suggest that the AtMGT gene family encodes a Mg(2+) transport system in higher plants.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.     

Effects of calcium and soluble cytoplasmic activator protein (calmodulin) on various states of (Ca2+ + Mg2+)-ATPase activity in isolated membranes of human red cells

(Ca2+ + Mg2+)-ATPase activity of red cells and their isolated membranes was investigated in the presence of various Ca2+ concentrations and cytoplasmic activator protein. Red cell ATPase activity was high at low Ca2+ concentrations, and low at moderate and high concentrations of Ca2+. In the case of isolated membranes, both low and moderate ca2+ concentrations produced higher (Ca2+ + Mg2+)-ATPase activity than high Ca2+ concentration. Membrane-free hemolysate containing soluble activator of (Ca2+ + Mg2+)-ATPase produced a significant increase in (Ca2+ + Mg2+)-ATPase activity only at low ca2+ concentration. Regardless of Ca2+ and activator concentrations, the enzyme activity in the membrane was lower than lysed red cells. The low level of (Ca2+ + Mg2+)-ATPase activity seen at high Ca2+ concentration can be augmented by lowering the Ca2+ concentration of EGTA in the assay medium. However, once the membrane was exposed to a high Ca2+ concentration, the activator could no longer exert it maximum stimulation at the low Ca2+ concentration brought about by addition of EGTA. This loss of activation was not attributable to the Ca2+-induced denaturation of activator protein but rather related to the alteration of (Ca2+ + Mg2+)-ATPase states in the membrane. On the basis of these data, it is suggested that only a small portion of (Ca2+ + Mg2+)-ATPase activity of isolated membranes can be stimulated by the soluble activator and that (ca2+ + Mg2+)ATPase most likely exists in various states depending upon ca2+ concentration and the presence of activator. The enzyme state exhibiting the high degree of stimulation by activator may undergo irreversible damage in the presence of high Ca2+ concentrations.     

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5‐bisphosphate signal on the plasma membrane and attenuates root hair elongation

Phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] serves as a subcellular signal on the plasma membrane, mediating various cell‐polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant‐unique plasma membrane protein with phosphoinositide‐binding activity, in PtdIns(4,5)P₂ signaling for root hair tip growth. The long‐root‐hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P₂‐producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter‐driven PCaP2–green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P₂ marker 2xCHERRY‐2xPH^PLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P₂ signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide‐binding domain PCaP2^N23, root hair‐specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2–GFP. Inducibly overexpressed PCaP2–GFP, but not its derivative lacking the PCaP2^N23 domain, replaced 2xCHERRY‐2xPH^PLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4‐64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2–GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P₂ signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P₂ and attenuates root hair elongation.     

The Ca2+‐binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer

Plasma membrane‐associated Ca²⁺‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca²⁺/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca²⁺. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca²⁺/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca²⁺. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2^G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca²⁺ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.