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.     

PIPKs are essential for rhizoid elongation and caulonemal cell development in the moss Physcomitrella patens

PtdIns‐4,5‐bisphosphate is a lipid messenger of eukaryotic cells that plays a critical role in processes such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels and nuclear signalling pathways. The enzymes responsible for the synthesis of PtdIns(4,5)P₂ are phosphatidylinositol phosphate kinases (PIPKs). The moss Physcomitrella patens contains two PIPKs, PpPIPK1 and PpPIPK2. To study their physiological role, both genes were disrupted by targeted homologous recombination and as a result mutant plants with lower PtdIns(4,5)P₂ levels were obtained. A strong phenotype for pipk1, but not for pipk2 single knockout lines, was obtained. The pipk1 knockout lines were impaired in rhizoid and caulonemal cell elongation, whereas pipk1‐2 double knockout lines showed dramatic defects in protonemal and gametophore morphology manifested by the absence of rapidly elongating caulonemal cells in the protonemal tissue, leafy gametophores with very short rhizoids, and loss of sporophyte production. pipk1 complemented by overexpression of PpPIPK1 fully restored the wild‐type phenotype whereas overexpression of the inactive PpPIPK1E885A did not. Overexpression of PpPIPK2 in the pipk1‐2 double knockout did not restore the wild‐type phenotype demonstrating that PpPIPK1 and PpPIPK2 are not functionally redundant. In vivo imaging of the cytoskeleton network revealed that the shortened caulonemal cells in the pipk1 mutants was the result of the absence of the apicobasal gradient of cortical F‐actin cables normally observed in wild‐type caulonemal cells. Our data indicate that both PpPIPKs play a crucial role in the development of the moss P. patens, and particularly in the regulation of tip growth.     

Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K7, PIP5K8, and PIP5K9 are redundantly involved in root growth adaptation to osmotic stress

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P₂), a signaling phospholipid critical for various cellular processes in eukaryotes. The Arabidopsis thaliana genome encodes 11 PIP5K genes. Of these, three type B PIP5K genes, PIP5K7, PIP5K8, and PIP5K9, constitute a subgroup highly conserved in land plants, suggesting that they retain a critical function shared by land plants. In this study, we comprehensively investigated the biological functions of the PIP5K7–9 subgroup genes. Reporter gene analyses revealed their preferential expression in meristematic and vascular tissues. Their YFP-fusion proteins localized primarily to the plasma membrane in root meristem epidermal cells. We selected a mutant line that was considered to be null for each gene. Under normal growth conditions, neither single mutants nor multiple mutants of any combination exhibited noticeable phenotypic changes. However, stress conditions with mannitol or NaCl suppressed main root growth and reduced proximal root meristem size to a greater extent in the pip5k7pip5k8pip5k9 triple mutant than in the wild type. In root meristem epidermal cells of the triple mutant, where plasma membrane localization of the PtdIns(4,5)P₂ marker P24Y is impaired to a large extent, brefeldin A body formation is retarded compared with the wild type under hyperosmotic stress. These results indicate that PIP5K7, PIP5K8, and PIP5K9 are not required under normal growth conditions, but are redundantly involved in root growth adaptation to hyperosmotic conditions, possibly through the PtdIns(4,5)P₂ function promoting plasma membrane recycling in root meristem cells.     

Arranged marriage in lipid signalling? The limited choices of PtdIns(4,5)P2 in finding the right partner

Inositol‐containing phospholipids (phosphoinositides, PIs) control numerous cellular processes in eukaryotic cells. For plants, a key involvement of PIs has been demonstrated in the regulation of membrane trafficking, cytoskeletal dynamics and in processes mediating the adaptation to changing environmental conditions. Phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P₂) mediates its cellular functions via binding to various alternative target proteins. Such downstream targets of PtdIns(4,5)P₂ are characterised by the possession of specific lipid‐binding domains, and binding of the PtdIns(4,5)P₂ ligand exerts effects on their activity or localisation. The large number of potential alternative binding partners – and associated cellular processes – raises the question how alternative or even contrapuntal effects of PtdIns(4,5)P₂ are orchestrated to enable cellular function. This article aims to provide an overview of recent insights and new views on how distinct functional pools of PtdIns(4,5)P₂ are generated and maintained. The emerging picture suggests that PtdIns(4,5)P₂ species containing different fatty acids influence the lateral mobility of the lipids in the membrane, possibly enabling specific interactions of PtdIns(4,5)P₂ pools with certain downstream targets. PtdIns(4,5)P₂ pools with certain functions might also be defined by protein–protein interactions of PI4P 5‐kinases, which pass PtdIns(4,5)P₂ only to certain downstream partners. Individually or in combination, PtdIns(4,5)P₂ species and specific protein–protein interactions of PI4P 5‐kinases might contribute to the channelling of PtdIns(4,5)P₂ signals towards specific functional effects. The dynamic nature of PI‐dependent signalling complexes with specific functions is an added challenge for future studies of plant PI signalling.     

The Arabidopsis Phosphatidylinositol Phosphate 5-Kinase PIP5K3 is a key regulator of root hair tip growth

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P2, that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.     

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin‐dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P₂ as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G‐actin unlike PIPKs from other organisms. PtdIns(4,5)P₂, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP‐tagged PH‐domain from PLCδ, which specifically binds PtdIns(4,5)P₂ in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.     

Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

Background  

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P ₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P ₂ also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P ₂ levels in living cells is critical to understanding the biology of PtdIns(4,5)P ₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P ₂ imaging tools.     

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P₂ effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P₂-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P₂. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P₂ in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P₂ in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P₂ functions to coordinate cytoskeletal dynamics and secretion.

The apical plasma membrane of pollen tubes contains nanodomains where the regulatory phospholipid PtdIns(4,5)P₂ exerts a stabilizing effect on the actin cytoskeleton.     

The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [³H-]phosphatidylinositol 4,5-biphosphate ([³H-]PtdIns(4,5)P ₂) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca²⁺,14 mM Na-cholate and 20 AM [³H-]PtdIns (4,5)P ₂ (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg^–1·2.5 min^–1. The addition of Mg²⁺ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na⁺ and at a low Ca²⁺ concentration (0.3 M), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀0.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg^–1·2.5 min^–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg^–1·2.5 min^–1). Unlike PtdIns(4,5)P ₂, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca²⁺ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP ₂ inositol bisphosphate (unidentified isomer) - InsP ₃ inositol trisphosphate (unidentified isomer) - InsP _x any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma     

Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement

A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.     

The recognition of membrane‐bound PtdIns3P by PX domains

Phox‐homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40^phox–PX domain and the SNX17–PX domain and their interaction with membrane‐bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane‐bound PtdIns3P and formed stable complexes. The interaction between the p40^phox–PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40^phox–PX–PtdIns3P complex that is available. The interaction between the SNX17–PX domain and PtdIns3P was similar to that observed in the p40^phox–PX–PtdIns3P complex; however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the β1–β2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2‐oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules. Proteins 2014; 82:2332–2342. © 2014 Wiley Periodicals, Inc.     

Cell biology and genetics of root hair formation inArabidopsis thaliana

Summary In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants ofArabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca²⁺ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation, to control of tip diameter and extent and polarity of elongation.Abbreviations [Ca²⁺]_c cytosolic calcium concentration - MT microtubule - PM plasma membrane - VRZ vesicle-rich zone - WT wild type Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway

The C2 domain is a targeting domain that responds to intracellular Ca²⁺ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P₂ specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca²⁺ specifically binds to the Ca²⁺-binding region, facilitating PtdIns(4,5)P₂ access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P₂ binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P₂ binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P₂ and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P₂ is hydrolyzed to generate diacylglycerol and IP₃, an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.     

PtdIns(4,5)P2 is not required for secretory granule docking

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co‐clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high‐resolution total internal reflection imaging of EGFP‐labeled PtdIns markers or syntaxin‐1 at secretory granule release sites in live insulin‐secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin‐1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin‐1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P₂) by recruitment of a 5′‐phosphatase strongly inhibited Ca²⁺‐dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin‐1. Cell permeabilization by α‐toxin or formaldehyde‐fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P₂ accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin‐1 at the release site.      

A mutant form of PTEN linked to autism

The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3‐position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism‐related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine‐bearing model membranes is 5.6‐fold enhanced in comparison to wild‐type PTEN. In contrast, we find that binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P₂) model membranes is 2.5‐fold decreased for the mutant PTEN in comparison to wild‐type PTEN. The structural change previously found for wild‐type PTEN upon interaction with PI(4,5)P₂, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN‐GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P₃ turnover, since in vivo studies show a reduced activity of the H93R PTEN‐GFP mutant. Because the interaction of PI(4,5)P₂ with PTEN's N‐terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N‐terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P₂ from inducing the conformational change that activates phosphatase activity.     

Phosphatidylinositol (4,5)-bisphosphate modulates Nox5 localization via an N-terminal polybasic region

Nox5, an EF-hand–containing reactive oxygen species (ROS)-generating NADPH oxidase, contains two conserved polybasic regions: one N-terminal (PBR-N), located between the fourth EF-hand and the first transmembrane region, and one C-terminal (PBR-C), between the first and second NADPH-binding subregions. Here, we show that phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂], a major phosphoinositide in plasma membrane, binds to human Nox5 causing Nox5 to localize from internal membranes to the plasma membrane. Enzymatic modulation of PtdIns(4,5)P₂ levels in intact cells altered cell surface localization of Nox5 in parallel with extracellular ROS generation. Mutations in PBR-N prevented PtdIns(4,5)P₂-dependent localization of Nox5 to the plasma membrane and decreased extracellular ROS production. A synthetic peptide corresponding to PBR-N bound to PtdIns(4,5)P₂, but not to PtdIns, whereas mutations in the PBR-N peptide abrogated the binding to PtdIns(4,5)P₂. Arginine-197 in PBR-N was a key residue to regulate subcellular localization of Nox5 and its interaction with PtdIns(4,5)P₂. In contrast, mutation in PBR-C did not affect localization. Thus, extracellular ROS production by Nox5 is modulated by PtdIns(4,5)P₂ by localizing Nox5 to the plasma membrane.     

Phosphatidylinositol 4,5-bisphosphate stimulates phosphorylation of the adaptor protein Shc by c-Src

The adaptor protein Shc was prepared as glutathione S-transferase fusion proteins (GST–Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of Shc has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584–592], effect of PtdIns(4,5)P2 on the phosphorylation of GST–Shc by c-Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST–Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. K_m for GST–Shc in the presence of 1 μM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST–Shc was inhibited by a GST–fusion protein containing the phosphotyrosine-binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c-Src through its binding to Shc.     

Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P₂ production at that location are uncharacterized, functions of PtdIns(4,5)P₂ in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P₂ production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P₂ production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P₂ via regulatory effects on membrane trafficking and/or apical pectin deposition.