Alternative complex formation of the Ca-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis

Intracellular release of calcium ions belongs to the earliest events in cellular stress perception. The molecular mechanisms integrating signals from different environmental cues and translating them into an optimized response are largely unknown. We report here the functional characterization of CIPK1, a protein kinase interacting strongly with the calcium sensors CBL1 and CBL9. Comparison of the expression patterns indicates that the three proteins execute their functions in the same tissues. Physical interaction of CIPK1 with CBL1 and CBL9 targets the kinase to the plasma membrane. We show that, similarly to loss of CBL9 function, mutation of either CBL1 or CIPK1 renders plants hypersensitive to osmotic stress. Remarkably, in contrast to the cbl1 mutant and similarly to the cbl9 mutant, loss of CIPK1 function impairs abscisic acid (ABA) responsiveness. We therefore suggest that, by alternative complex formation with either CBL1 or CBL9, the kinase CIPK1 represents a convergence point for ABA-dependent and ABA-independent stress responses. Based on our genetic, physiological and protein-protein interaction data, we propose a general model for information processing in calcium-regulated signalling networks.     

Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network

Plant development and reproduction depend on a precise recognition of environmental conditions and the integration of this information with endogenous metabolic and developmental cues. Calcium ions have been firmly established as ubiquitous second messengers functioning in these processes. Calcium signal deciphering and signal-response coupling often involve calcium-binding proteins as responders or relays in this information flow. Here we review the calcineurin B-like protein (CBL) calcium sensor/CBL-interacting protein kinase (CIPK) network as a newly emerging signaling system mediating a complex array of environmental stimuli. We focus particularly on the mechanisms generating signaling specificity. Moreover, we emphasize the functional implications that are emerging from the analyses of CBL and CIPK loss-of-function mutants.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.     

The occurrence of 'bulbs', a complex configuration of the vacuolar membrane, is affected by mutations of vacuolar SNARE and phospholipase in Arabidopsis

The plant vacuole fulfills a variety of functions, and is essential for plant growth and development. We previously identified complex and mobile structures on the continuous vacuolar membrane, which we refer to as 'bulbs'. To ascertain their biological significance and function, we searched for markers associated with bulbs, and mutants that show abnormalities with respect to bulbs. We observed bulb-like structures after expression of non-membranous proteins as well as the functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecules VAM3 and VTI11. Bulbs are formed in more tissues than previously reported, including flowering organs, suspension culture cells, endodermal cells in the flowering stem, and at very early stages of seed germination. Using existing and newly developed marker lines, we found that the frequency of bulb occurrence is significantly decreased in multiple shoot gravitropism (sgr) mutants, which are known to have a defect in vacuolar membrane properties in endodermal cells. Based on results with new marker lines, which enabled us to observe the process of bulb biogenesis, and analysis of the phenotypes of these mutants, we propose multiple mechanisms for bulb formation, one of which may be that used for formation of transvacuolar strands.     

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3^(?1–270) expressed in S. pombe avt3? cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3⁺ but was not completely rescued by the expression of avt3^(?1–270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.     

Vba4p,a vacuolar membrane protein,is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.     

Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex

Potassium (K(+)) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K(+) channels remain poorly understood. Here, we show that the calcium (Ca(2+)) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K(+) channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca(2+)-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca(2+) sensor modulates K(+) channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.     

Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A-stimulated platelets

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca²⁺. This effect was due to two different processes: Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca²⁺ from internal storage sites occurred sooner than the opening of plasma membrane Ca²⁺ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca²⁺ concentration was antagonized and reversed by methyl ∝-D -mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl–concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl–concanavalin A, per se, was unable to induce Ca²⁺ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca²⁺ from the same internal stores from which Ca²⁺ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca²⁺ release after stimulation of platelets with Con A, Con A was able to cause a full discharge of Ca²⁺ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.     

A protein phosphatase 2A catalytic subunit is a negative regulator of abscisic acid signalling

The key regulatory role of abscisic acid (ABA) in many physiological processes in plants is well established. However, compared with other plant hormones, the molecular mechanisms underlying ABA signalling are poorly characterized. In this work, a specific catalytic subunit of protein phosphatase 2A (PP2Ac-2) has been identified as a component of the signalling pathway that represses responses to ABA. A loss-of-function pp2ac-2 mutant is hypersensitive to ABA. Moreover, pp2ac-2 plants have altered responses in developmental and environmental processes that are mediated by ABA, such as primary and lateral root development, seed germination and responses to drought and high salt and sugar stresses. Conversely, transgenic plants overexpressing PP2Ac-2 are less sensitive to ABA than wild type, a phenotype that is manifested in all the above-mentioned physiological processes. DNA microarray hybridization experiments reveal that PP2Ac-2 is negatively involved in ABA responses through regulation of ABA-dependent gene expression. Moreover, the results obtained indicate that ABA antagonistically regulates PP2Ac-2 expression and PP2Ac-2 activity thus allowing plant sensitivity to the hormone to be reset after induction. Phenotypic, genetic and gene expression data strongly suggest that PP2Ac-2 is a negative regulator of the ABA pathway. Activity of protein phosphatase 2A thus emerges as a key element in the control of ABA signalling.     

An improved method for the direct chromatographic resolution of abscisic acid enantiomers

An improved method for the direct chromatographic resolution of abscisic acid enantiomers using the commercially available Whelk-O 1 chiral stationary phase is presented. Previously reported strategies utilized in the chromatographic separation of abscisic acid enantiomers are summarized, and conditions for analytical and semipreparative separations using the Whelk-O 1 chiral stationary phase are described. This method offers the advantages of rapid analysis time and greatly increased capacity, allowing the resolution of more than 6 mg in 25 min using an analytical column (4.6 mm i.d. × 25 cm length). © 1993 Wiley-Liss, Inc.     

Association of Arabidopsis type-II ROPs with the plasma membrane requires a conserved C-terminal sequence motif and a proximal polybasic domain

Plant ROPs (or RACs) are soluble Ras-related small GTPases that are attached to cell membranes by virtue of the post-translational lipid modifications of prenylation and S-acylation. ROPs (RACs) are subdivided into two major subgroups called type-I and type-II. Whereas type-I ROPs terminate with a conserved CaaL box and undergo prenylation, type-II ROPs undergo S-acylation on two or three C-terminal cysteines. In the present work we determined the sequence requirement for association of Arabidopsis type-II ROPs with the plasma membrane. We identified a conserved sequence motif, designated the GC-CG box, in which the modified cysteines are flanked by glycines. The GC-CG box cysteines are separated by five to six mostly non-polar residues. Deletion of this sequence or the introduction of mutations that change its nature disrupted the association of ROPs with the membrane. Mutations that changed the GC-CG box glycines to alanines also interfered with membrane association. Deletion of a polybasic domain proximal to the GC-CG box disrupted the plasma membrane association of AtROP10. A green fluorescent protein fusion protein containing the C-terminal 25 residues of AtROP10, including its polybasic domain and GC-CG box, was primarily associated with the plasma membrane but a similar fusion protein lacking the polybasic domain was exclusively localized in the soluble fraction. These data provide evidence for the minimal sequence required for plasma membrane association of type-II ROPs in Arabidopsis and other plant species.     

Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum

Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducing channel in the parasitophorous vacoular membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a 'translocation competent' state prior to membrane passage.     

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.     

Vanadate-activated calcium influx in A431 cells is dependent on the plasma membrane potential

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two- to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down-regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium-independent and is not activated by depolarization in high KCl. On the contrary, vanadate-stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about -65 to -30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.     

The amino-terminal region of the Escherichia coli T-protein of the glycine cleavage system is essential for proper association with H-protein.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. Our previous work on Escherichia coli T-protein (ET) showed that the lack of the N-terminal 16 residues caused a loss of catalytic activity [Okamura-Ikeda, K., Ohmura, Y., Fujiwara, K. and Motokawa, Y. (1993) Eur. J. Biochem. 216, 539-548]. To define the role of the N-terminal region of ET, a series of deletion mutants were constructed by site-directed mutagenesis and expressed in E. coli. Deletions of the N-terminal 4, 7 and 11 residues led to reduction in the activity to 42, 9 and 4%, respectively, relative to the wild-type enzyme (wtET). The mutant with 7-residue deletion (ETDelta7) was purified and analyzed. ETDelta7 exhibited a marked increase in Km (25-fold) for E. coli H-protein (EH) accompanied by a 10-fold decrease in kcat compared with wtET, indicating the importance of the N-terminal region in the interaction with EH. The role of this region in the ET-EH interaction was investigated by cross-linking of wtET-EH or ETDelta7-EH complex with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker, in the presence of folate substrates. The resulting tripartite cross-linked products were cleaved with lysylendopeptidase and V8 protease. After purification by reversed-phase HPLC, the cross-linked peptides were subjected to Edman sequencing. An intramolecular cross-linking between Asp34 and Lys216 of wtET which was not observed in wtET alone and an intermolecular cross-linking between Lys288 of wtET and Asp-43 of EH were identified. In contrast, no such cross-linking was detected from the cross-linked product of ETDelta7. These results suggest that EH, when it interacts with ET, causes a change in conformation of ET and that the N-terminal region of ET is essential for the conformational change leading to the proper interaction with EH.     

The plasma membrane calcium ATPase MCA-3 is required for clathrin-mediated endocytosis in scavenger cells of Caenorhabditis elegans

Plasma membrane Ca2+ ATPases (PMCAs) maintain proper intracellular Ca2+ levels by extruding Ca2+ from the cytosol. PMCA genes and splice forms are expressed in tissue-specific patterns in vertebrates, suggesting that these isoforms may regulate specific biological processes. However, knockout mutants die as embryos or undergo cell death; thus, it is unclear whether other cell processes utilize PMCAs or whether these pumps are largely committed to the control of toxic levels of calcium. Here, we analyze the role of the PMCA gene, mca-3, in Caenorhabditis elegans. We report that partial loss-of-function mutations disrupt clathrin-mediated endocytosis in a class of scavenger cells called coelomocytes. Moreover, components of early endocytic machinery are mislocalized in mca-3 mutants, including phosphatidylinositol-4,5-bisphosphate, clathrin and the Eps15 homology (EH) domain protein RME-1. This defect in endocytosis in the coelomocytes can be reversed by lowering calcium. Together, these data support a function for PMCAs in the regulation of endocytosis in the C. elegans coelomocytes. In addition, they suggest that endocytosis can be blocked by high calcium levels.