Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kalpha1 and AtPI4Kbeta1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kalpha1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kalpha1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kalpha1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kalpha1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kalpha1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4Kbeta1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kalpha1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kbeta1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kalpha1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kalpha1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.     

Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase beta1 to the actin cytoskeleton

The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kbeta1 to the cytoskeleton. Recruitment of AtPI4Kbeta1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane.     

Multiple roles for phosphatidylinositol 4-kinase in biosynthetic transport in polarized Madin-Darby canine kidney cells.

Phosphatidylinositols (PI) play important roles in regulating numerous cellular processes including cytoskeletal organization and membrane trafficking. The control of PI metabolism by phosphatidylinositol kinases has been the subject of extensive investigation; however, little is known about how phosphatidylinositol kinases regulate traffic in polarized epithelial cells. Because phosphatidylinositol 4-kinase (PI4K)-mediated phosphatidylinositol 4-phosphate (PI(4)P) production has been suggested to regulate biosynthetic traffic in yeast and mammalian cells, we have examined the role of PI4Kbeta in protein delivery in polarized MDCK cells, at different levels of the biosynthetic pathway. Expression of wild type PI4Kbeta had no effect on the rate of transport of influenza hemagglutinin (HA) through the Golgi complex, but inhibited the rate of trans-Golgi network (TGN)-to-cell surface delivery of this protein. By contrast, expression of dominant-negative, kinase-dead PI4Kbeta (PI4Kbeta(D656A)) inhibited intra-Golgi transport but stimulated TGN-to-cell surface delivery of HA. Moreover, expression of PI4Kbeta(D656A) significantly increased the solubility in cold Triton X-100 of HA staged in the TGN, suggesting that altered association of HA with lipid rafts may be responsible for the enhanced transport rate. Both wild type and kinase-dead PI4Kbeta inhibited basolateral delivery of vesicular stomatitis virus G protein, suggesting an effector function for PI4Kbeta in the regulation of basolateral traffic. Thus, by contrast with the observed requirement for PI4Kbeta activity and PI(4)P for efficient transport in yeast, our data suggest that changes in PI(4)P levels can stimulate and inhibit Golgi to cell surface delivery in mammalian cells.     

Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.     

Eat in or take away? How phosphatidylinositol 4-kinases feed the phospholipase C pathway with substrate

Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first step in the synthesis of phosphoinositide pools hydrolysed by phosphoinositide-dependent phospholipase C (PI-PLC) and thus constitute a potential key regulation point of this pathway. Twelve putative PI4K isoforms, divided as type-II (AtPI4KIIγ1-8) and type-III PI4Ks (AtPI4KIIIα1-2 and AtPI4KIIIβ1-2), have been identified in Arabidopsis genome. By a combination of pharmalogical and genetic approaches we recently evidenced that AtPI4KIIIβ1 and AtPI4KIIIβ2 contribute to supply PI-PLC with substrate and that AtPI4KIIIα1 is probably also involved in this process. Given the current knowledge on PI-PLC and type-III PI4Ks localization in plant cells it raises the question whether type-III PI4Ks produce phosphatidylinositol 4-phosphate at the site of its consumption by the PI-PLC pathway. We therefore discuss the spatial organization of substrate supply to PI-PLC in plant cells with reference to recent data evidenced in mammalian cells.     

Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta regulate IgE receptor-triggered exocytosis in cultured mast cells

We examined the possible occurrence and function of neuronal Ca(2+) sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase beta (PI4Kbeta) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kbeta, increases IgE receptor (FcepsilonRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), and stimulates FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kbeta reduces PI4Kbeta activity, decreases FcepsilonRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kbeta, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P(2) that serves as substrate for the generation of lipid-derived second messengers in FcepsilonRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kbeta and positive regulation of its activity.     

Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta stimulate extracellular signal-regulated kinase 1/2 signaling by accelerating recycling through the endocytic recycling compartment

We demonstrate that recycling through the endocytic recycling compartment (ERC) is an essential step in Fc epsilonRI-induced activation of extracellular signal-regulated kinase (ERK)1/2. We show that ERK1/2 acquires perinuclear localization and colocalizes with Rab 11 and internalized transferrin in Fc epsilonRI-activated cells. Moreover, a close correlation exists between the amount of ERC-localized ERK1/2 and the amount of phospho-ERK1/2 that resides in the nucleus. We further show that by activating phosphatidylinositol 4-kinase beta (PI4Kbeta) and increasing the cellular level of phosphatidylinositol(4) phosphate, neuronal calcium sensor-1 (NCS-1), a calmodulin-related protein, stimulates recycling and thereby enhances Fc epsilonRI-triggered activation and nuclear translocation of ERK1/2. Conversely, NCS-1 short hairpin RNA, a kinase dead (KD) mutant of PI4Kbeta (KD-PI4Kbeta), the pleckstrin homology (PH) domain of FAPP1 as well as RNA interference of synaptotagmin IX or monensin, which inhibit export from the ERC, abrogate Fc epsilonRI-induced activation of ERK1/2. Consistently, NCS-1 also enhances, whereas both KD-PI4Kbeta and FAPP1-PH domain inhibit, Fc epsilonRI-induced release of arachidonic acid/metabolites, a downstream target of ERK1/2 in mast cells. Together, our results demonstrate a novel role for NCS-1 and PI4Kbeta in regulating ERK1/2 signaling and inflammatory reactions in mast cells. Our results further identify the ERC as a crucial determinant in controlling ERK1/2 signaling.     

The Highly Charged Region of Plant β-type Phosphatidylinositol 4-kinase is Involved in Membrane Targeting and Phospholipid Binding

In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The α-type PI4Ks (~220 kDa) contain a PH domain, which is lacking in β-type PI4Ks (~120 kDa). β-Type PI4Ks, exemplified by Arabidopsis AtPI4Kβ and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant β-type PI4Ks at present. The PPC region has a length of ~300 amino acids and harboring 11 (AtPI4Kβ) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based “Sequence of Membrane-Targeting Detection” system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1–8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kβ was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P₂ in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant β-type PI4Ks. The nucleotide sequences reported in this paper have been submitted to the GenBank^TM/EMBL Data Bank under accession number AY536061 (highly charged region of OsPI4K2) and AJ277791 (partial cDNA of OsPI4K2). Ying Lou and Hui Ma: These authors contributed equally     

Role of Arabidopsis AtPI4Kγ3, a type II phosphoinositide 4‐kinase,in abiotic stress responses and floral transition

Phosphoinositides (PIs) are essential metabolites which are generated by various lipid kinases and rapidly respond to a variety of environmental stimuli in eukaryotes. One of the precursors of important regulatory PIs, phosphatidylinositol (PtdIn) 4‐phosphate, is synthesized by PtdIns 4‐kinases (PI4K). Despite its wide distribution in eukaryotes, its role in plants remains largely unknown. Here, we show that the activity of AtPI4Kγ3 gene, an Arabidopsis (Arabidopsis thaliana) type II PtdIn 4‐kinase, is regulated by DNA demethylation and some abiotic stresses. AtPI4Kγ3 is targeted to the nucleus and selectively bounds to a few PtdIns. It possessed autophosphorylation activity but unexpectedly had no detectable lipid kinase activity. Overexpression of AtPI4Kγ3 revealed enhanced tolerance to high salinity or ABA along with inducible expression of a host of stress‐responsive genes and an optimal accumulation of reactive oxygen species. Furthermore, overexpressed AtPI4Kγ3 augmented the salt tolerance of bzip60 mutants. The ubiquitin‐like domain of AtPI4Kγ3 is demonstrated to be essential for salt stress tolerance. Besides, AtPI4Kγ3‐overexpressed plants showed a late‐flowering phenotype, which was caused by the regulation of some flowering pathway integrators. In all, our results indicate that AtPI4Kγ3 is necessary for reinforcement of plant response to abiotic stresses and delay of the floral transition.     

Interaction of neuronal calcium sensor-1 and ADP-ribosylation factor 1 allows bidirectional control of phosphatidylinositol 4-kinase beta and trans-Golgi network-plasma membrane traffic

We have identified a novel Ca(2+)-dependent interaction between neuronal calcium sensor-1 (NCS-1) and the GTPase ARF1. Both of these proteins are localized to the Golgi complex, and both regulate phosphatidylinositol 4-kinase IIIbeta (PI(4)Kbeta). Spatial and temporal control of phosphatidylinositol 4-phosphate levels through activation of PI(4)Kbeta is important for the recruitment of trafficking complexes to the trans-Golgi network (TGN) and vesicular traffic from this organelle. The NCS-1-ARF1 interaction and its specificity have been demonstrated through in vitro binding assays, in vitro enzyme assay, and through functional cellular assays. We show that NCS-1 can exert bidirectional effects to activate PI(4)Kbeta on its own or inhibit the activation by ARF1. NCS-1 was shown to modulate the effects of expression of ARF mutants that disrupt Golgi morphology and to recruit GDP-loaded ARF to the Golgi complex in a Ca(2+)-dependent manner. We demonstrate antagonist effects of NCS-1 and ARF on constitutive and regulated exocytosis. The NCS-1-ARF1 interaction provides evidence for functional cross-talk between Ca(2+)-dependent and ARF-dependent pathways in TGN to plasma membrane traffic.     

Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.     

Characterization of recombinant phosphatidylinositol 4-kinase beta reveals auto- and heterophosphorylation of the enzyme

Phosphatidylinositol (PI) 4-kinases catalyze the synthesis of PI 4-phosphate, an important intermediate for the synthesis of membrane polyphosphoinositides, regulators of multiple cellular functions. Two mammalian PI 4-kinases have been cloned, a 230-kDa enzyme (alpha-form) and a 110-kDa (beta-form), both of which are inhibited by >0.1 microm concentrations of the PI 3-kinase inhibitor, wortmannin (WT). In the present study, we created a glutathione S-transferase-PI4Kbeta fusion protein for expression in Escherichia coli. The purified protein was biologically active and phosphorylated PI in its 4-position with WT sensitivity and kinetic parameters that were identical to those of purified bovine brain PI4Kbeta. In addition to its lipid kinase activity, the enzyme exhibited autophosphorylation that was enhanced by Mn(2+) ions and inhibited by WT and another PI 3-kinase inhibitor, LY 294002. The recombinant protein was unable to transphosphorylate, but its isolated C-terminal catalytic domain still displayed autophosphorylation, suggesting that the autophosphorylation site resides within the C-terminal catalytic domain of the protein and is held in position by intramolecular interactions. Autophosphorylation inhibited subsequent lipid kinase activity, which was reversed upon dephosphorylation, by protein phosphatases, PP1 and PP2A(1), suggesting that it may represent a regulatory mechanism for the enzyme. Phosphorylation of endogenous or overexpressed PI4Kbeta was also observed in COS-7 cells; however, the in vivo phosphorylation of the expressed protein was only partially inhibited by WT and also occurred in a catalytically inactive form of the enzyme, indicating the presence of additional phosphorylation site(s). Successful bacterial expression of PI4Kbeta should aid research on the structure-function relationships of this protein as well as of other, structurally related enzymes.     

Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.     

Type II phosphatidylinositol 4-kinases promote Listeria monocytogenes entry into target cells

Interaction of the Listeria surface protein InlB with the hepatocyte growth factor receptor Met activates signalling events that trigger bacterial internalization into mammalian epithelial cells. We show here that purified phagosomes containing InlB-coated beads display type II phosphatidylinositol 4-kinase (PI4K) activity. In human epithelial HeLa cells, both PI4KIIalpha and PI4KIIbeta isoforms are corecruited with Met around InlB-coated beads or wild-type Listeria during the early steps of internalization, and phosphatidylinositol 4-phosphate [PI(4)P] is detected at the entry site. We demonstrate that PI4KIIalpha or PI4KIIbeta knockdown, but not type III PI4Kbeta knockdown, inhibits Listeria internalization. Production of PI(4)P derivatives such as phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] upon InlB stimulation is not affected by PI4KIIalpha or beta knockdown, suggesting that these phosphoinositides are generated by a type III PI4K. Strikingly, knockdown of the PI(4)P ligand and clathrin adaptor AP-1 strongly inhibits bacterial entry. Together, our results reveal a yet non-described role for type II PI4Ks in phagocytosis.     

Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.     

Cloning of Arabidopsis thaliana phosphatidylinositol synthase and functional expression in the yeast pis mutant

It is believed that phosphatidylinositol (PI) metabolism plays a central role in signalling pathways in both animals and higher plants. PI is synthesized from CDP-diacylglycerol (CDP-DG) and myo-inositol by phosphatidylinositol synthase (PI synthase, EC 2.7.8.11). Here we report the identification of a plant cDNA (AtPIS1) encoding a 26 kDa PI synthase from Arabidopsis thaliana. The plant enzyme as deduced from its cDNA sequence shares 35–41% identical amino acids with PI synthases from Saccharomyces cerevisiae and mammals. AtPIS1 functionally complements a mutant of S. cerevisiae with a lesion in PI synthase, and recombinant AtPIS1 protein present in yeast membranes strongly depends on the two principal substrates, myo-inositol and CDP-DG, and requires Mg²⁺ ions for full activity.     

A role for the RabA4b effector protein PI-4Kbeta1 in polarized expansion of root hair cells in Arabidopsis thaliana

The RabA4b GTPase labels a novel, trans-Golgi network compartment displaying a developmentally regulated polar distribution in growing Arabidopsis thaliana root hair cells. GTP bound RabA4b selectively recruits the plant phosphatidylinositol 4-OH kinase, PI-4Kbeta1, but not members of other PI-4K families. PI-4Kbeta1 colocalizes with RabA4b on tip-localized membranes in growing root hairs, and mutant plants in which both the PI-4Kbeta1 and -4Kbeta2 genes are disrupted display aberrant root hair morphologies. PI-4Kbeta1 interacts with RabA4b through a novel homology domain, specific to eukaryotic type IIIbeta PI-4Ks, and PI-4Kbeta1 also interacts with a Ca2+ sensor, AtCBL1, through its NH2 terminus. We propose that RabA4b recruitment of PI-4Kbeta1 results in Ca2+-dependent generation of PI-4P on this compartment, providing a link between Ca2+ and PI-4,5P2-dependent signals during the polarized secretion of cell wall components in tip-growing root hair cells.     

Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex

Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.