José Mulet

A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter

ABSTRACT The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes fromSaccharomyces cerevisiae, HAL4/SAT4 andHAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.

Yeast putative transcription factors involved in salt tolerance

Four putative yeast transcription factors (Hal6–9p) have been identified which upon overexpression in multicopy plasmids increase sodium and lithium tolerance. This effect is mediated, at least in part, by increased expression of the Ena1p Na+/Li+ extrusion pump. Hal6p and Hal7p are bZIP proteins and their gene disruptions affected neither salt tolerance nor ENA1 expression. Hal8p and Hal9p are putative zinc fingers and their gene disruptions decreased both salt tolerance and ENA1 expression. Therefore, Hal8p and Hal9p, but not Hal6p and Hal7p, qualify as transcriptional activators of ENA1 under physiological conditions. Hal8p seems to mediate the calcineurin‐dependent part of ENA1 expression.

The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression

The yeast Ppz protein phosphatases and the Hal3p inhibitory subunit are important determinants of salt tolerance, cell wall integrity and cell cycle progression. We present several lines of evidence showing that these disparate phenotypes are connected by the fact that Ppz regulates K+ transport. First, salt tolerance, cell wall integrity and cell cycle phenotypes of Ppz mutants are dependent on the Trk K+ transporters. Secondly, Ppz mutants exhibit altered activity of the Trk system, as measured by rubidium uptake. Thirdly, Ppz mutants exhibit altered intracellular K+ and pH, as expected from H+ efflux providing electrical balance during K+ uptake. Our unifying picture of Ppz phenotypes contends that activation of Trk by decreased Ppz activity results in plasma membrane depolarization (reducing uptake of toxic cations), increased intracellular K+ and turgor (compromising cell integrity), and increased intracellular pH (augmenting the expression of pH‐regulated genes and facilitating α‐factor recovery). In addition to providing a coherent explanation for all Ppz‐dependent phenotypes, our results provide evidence for a causal relationship between intracellular cation homeostasis and a potential cell cycle checkpoint.

Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Deficiency Leads to Altered Root Development and Affects the Sugar and Amino Acid Balance in Arabidopsis

Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD+ to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

Mutual antagonism of target of rapamycin and calcineurin signaling.

Growth and stress are generally incompatible states. Stressed cells adapt to an insult by restraining growth, and conversely, growing cells keep stress responses at bay. This is evident in many physiological settings, including for example, the effect of stress on the immune or nervous system, but the underlying signaling mechanisms mediating such mutual antagonism are poorly understood. In eukaryotes, a central activator of cell growth is the protein kinase target of rapamycin (TOR) and its namesake signaling network. Calcineurin is a conserved, Ca2+/calmodulin-dependent protein phosphatase and target of the immunosuppressant FK506 (tacrolimus) that is activated in yeast during stress to promote cell survival. Here we show yeast mutants defective for TOR complex 2 (TORC2) or the essential homologous TORC2 effectors, SLM1 and SLM2, exhibited constitutive activation of calcineurin-dependent transcription and actin depolarization. Conversely, cells defective in calcineurin exhibited SLM1 hyperphosphorylation and enhanced interaction between TORC2 and SLM1. Furthermore, a mutant SLM1 protein (SLM1ΔC14) lacking a sequence related to the consensus calcineurin docking site (PxIxIT) was insensitive to calcineurin, and SLM1ΔC14 slm2 mutant cells were hypersensitive to oxidative stress. Thus, TORC2-SLM signaling negatively regulates calcineurin, and calcineurin negatively regulates TORC2-SLM. These findings provide a molecular basis for the mutual antagonism of growth and stress.

A role for the non‐phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport

The Snf1/AMP‐activated protein kinases play a key role in stress responses of eukaryotic cells. In the yeast Saccharomyces cerevisiae Snf1 is regulated by glucose depletion, which triggers its phosphorylation at Thr210 and concomitant increase in activity. Activated yeast Snf1 is required for the metabolic changes allowing starvation tolerance and utilization of alternative carbon sources. We now report a function for the non‐activated form of Snf1: the regulation of the Trk high‐affinity potassium transporter, encoded by the TRK1 and TRK2 genes. A snf1Δ strain is hypersensitive in high‐glucose medium to different toxic cations, suggesting a hyperpolarization of the plasma membrane driving increased cation uptake. This phenotype is suppressed by the TRK1 and HAL5 genes in high‐copy number consistent with a defect in K+ uptake mediated by the Trk system. Accordingly, Rb+ uptake and intracellular K+ measurements indicate that snf1Δ is unable to fully activate K+ import. Genetic analysis suggests that the weak kinase activity of the non‐phosphorylated form of Snf1 activates Trk in glucose‐metabolizing yeast cells. The effect of Snf1 on Trk is probably indirect and could be mediated by the Sip4 transcriptional activator.

Potentiation of human α4β2 neuronal nicotinic receptors by a Flustra foliacea metabolite

Abstract The effects of various Flustra foliacea metabolites on different types of human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated. Whereas most of the compounds tested had a small blocking effect, one of them, deformylflustrabromine, selectively increased the current obtained in α4β2 receptors when co-applied with acetylcholine (ACh). The current increase was reversible and concentration-dependent. This potentiating effect was still present at saturating concentrations of acetylcholine, and no changes in single-channel conductance or reversal potential were observed, thus suggesting a modification in the gating of α4β2 receptors. Dwell time analysis of single channel records indicates that the mechanism of action of deformylflustrabromine could be both an increase of the opening rate constant and a decrease of the closing rate constant on α4β2 receptors. Thus, deformylflustrabromine may constitute an excellent starting point for the future development of related agents able to potentiate human neuronal nicotinic receptor function.

Charged Amino Acids of the N-terminal Domain Are Involved in Coupling Binding and Gating in α7 Nicotinic Receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the α7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.

The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter.

Protein kinases dedicated to the phosphorylation of SR proteins have been implicated in the processing and nuclear export of mRNAs. Here we demonstrate in Saccharomyces cerevisiae their participation in cation homeostasis. A null mutant of the single yeast SR protein kinase Sky1p is viable but exhibits increased tolerance to diverse toxic cations such as Na(+), Li(+), spermine, tetramethylammonium, hygromycin B and Mn(2+). This pleiotropic phenotype correlates with reduced accumulation of cations, suggesting a decrease in membrane electrical potential. Genetic analysis and Rb(+) uptake measurements indicate that Sky1p modulates Trk1,2, the high-affinity K(+) uptake system of yeast and a major determinant of membrane potential.

C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis

Subcellular localization of abscisic acid receptors as peripheral proteins in the plasma membrane is mediated in a calcium-dependent manner by C2-domain abscisic acid-related proteins. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.