Blanca Garciadeblas

A novel P‐type ATPase from yeast involved in sodium transport

The gene ENA1 was cloned by its ability to complement the Li+ sensitivity of a low Li+‐efflux strain. The nucleotide sequence of the cloned DNA fragment showed that there are two almost identical genes in tandem, and predicts that they encode P‐ATPases. Disruption of both genes originated a strain defective in Na+ and Li+ effluxes, and sensitive to Na+, to Li+ and to alkaline pH. By transformation with ENA1 the defective effluxes and tolerances were repaired.

Conservation of the Salt Overly Sensitive Pathway in Rice

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.

Inventory and Functional Characterization of the HAK Potassium Transporters of Rice

Plants take up large amounts of K+ from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K+from the soil solution to its final destination is mediated by channels and transporters. To better understand K+ movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativacv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1–17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K+ influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K+ influx in yeast and in roots of K+-starved rice seedlings demonstrated the involvement of HAK transporters in root K+ uptake. We discuss that all HAK transporters may mediate K+ transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.

Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae

SummaryThe ENA2 gene encoding a P-type ATPase involved in Na+ and Li+ effluxes in Saccharomyces cerevisiae has been isolated. The putative protein encoded by ENA2 differs only in thirteen amino acids from the protein encoded by ENA1/PMR2. However, ENA2 has a very low level of expression and for this reason did not confer significant Li+ tolerance on a Li+ sensitive strain. ENA1 and ENA2 are the first two units of a tandem array of four highly homologous genes with probably homologous functions.

Potassium- or sodium-efflux ATPase, a key enzyme in the evolution of fungi.

Potassium is the most abundant cation in cells. Therefore, plant-associated fungi and intracellular parasites are permanently or circumstantially exposed to high K(+) and must avoid excessive K(+) accumulation activating K(+) efflux systems. Because high K(+) and high pH are compatible in natural environments, free-living organisms cannot keep a permanent transmembrane DeltapH and cannot rely only on K(+)/H(+) antiporters, as do mitochondria. This study shows that the Schizosaccharomyces pombe CTA3 is a K(+)-efflux ATPase, and that other fungi are furnished with Na(+)-efflux ATPases, which also pump Na(+). All these fungal ATPases, including those pumping only Na(+), form a phylogenetic group, IID or ENA, among P-type ATPases. By searching in databases and partial cloning of ENA genes in species of Zygomycetes and Basidiomycetes, the authors conclude that probably all fungi have these genes. This study indicates that fungal K(+)- or Na(+)-ATPases evolved from an ancestral K(+)-ATPase, through processes of gene duplication. In yeast hemiascomycetes these duplications have occurred recently and produced bifunctional ATPases, whereas in Neurospora, and probably in other euascomycetes, they occurred earlier in evolution and produced specialized ATPases. In Schizosaccharomyces, adaptation to Na(+) did not involve the duplication of the K(+)-ATPase and thus it retains an enzyme which is probably close to the original one. The parasites Leishmania and Trypanosoma have ATPases phylogenetically related to fungal K(+)-ATPases, which are probably functional homologues of the fungal enzymes.

Molecular cloning of the calcium and sodium ATPases in Neurospora crassa

Using PCR, reverse transcription‐PCR (RT‐PCR) and colony hybridization in a genomic library, we isolated six genes which encode type II P‐type ATPases in Neurospora crassa. The six full‐length cDNAs were cloned in a yeast expression vector and transformed into Saccharomyces cerevisiae null Ca2+‐ or Na+‐ATPase mutants. Three cDNAs suppressed the defect of the Ca2+ mutant and two of these protected from Mn2+ toxicity. One cDNA suppressed the defect of the Na+ mutant and two cDNAs were not functional in S. cerevisiae. The expression of the transcripts of the six genes in the presence of Ca2+, Na+, high pH or supporting an osmotic shock indicated that, with the exception of one of the Ca2+‐ATPases, the main function of the cloned ATPases is the adaptation to stress conditions. The relationship between the cloned fungal Ca2+‐ and Na+‐ATPases and plant type II P‐ATPases is discussed.

Novel P-Type ATPases Mediate High-Affinity Potassium or Sodium Uptake in Fungi

ABSTRACT Fungi have an absolute requirement for K+, but K+ may be partially replaced by Na+. Na+ uptake in Ustilago maydis and Pichia sorbitophila was found to exhibit a fast rate, low Km, and apparent independence of the membrane potential. Searches of sequences with similarity to P-type ATPases in databases allowed us to identify three genes in these species, Umacu1, Umacu2, and PsACU1, that could encode P-type ATPases of a novel type. Deletion of the acu1 and acu2 genes proved that they encoded the transporters that mediated the high-affinity Na+ uptake of U. maydis. Heterologous expressions of the Umacu2 gene in K+ transport mutants of Saccharomyces cerevisiae and transport studies in the single and double Δacu1 and Δacu2 mutants of U. maydis revealed that the acu1 and acu2 genes encode transporters that mediated high-affinity K+ uptake in addition to Na+ uptake. Other fungi also have genes or pseudogenes whose translated sequences show high similarity to the ACU proteins of U. maydis and P. sorbitophila. In the phylogenetic tree of P-type ATPases all the identified ACU ATPases define a new cluster, which shows the lowest divergence with type IIC, animal Na+,K+-ATPases. The fungal high-affinity Na+ uptake mediated by ACU ATPases is functionally identical to the uptake that is mediated by some plant HKT transporters.

Plant cells express several stress calcium ATPases but apparently no sodium ATPase

The existence of plant Na+-ATPases has been investigated in barley (Hordeum vulgare) and in the seagrass Cymodocea nodosa, by a combination of RT–PCR and flux approaches. Systematic RT–PCR amplifications were carried out in mRNA preparations of barley roots exposed to Na+ or of Cymodocea leaves, using degenerate primers that can amplify all known plant and fungal Na+- and Ca2+-ATPases and animal Na+,K+-ATPases. This allowed the amplification of fourteen different cDNAs that could encode P-type ATPases. A phylogenetic analysis showed that none of these ATPases belongs to the ENA type, in which all fungal Na+-ATPases cluster, or to the animal Na+,K+-ATPase type, and that all cluster with known plant and fungal Ca2+-ATPases. Expression analysis of the barley transcripts indicates that the expressions of all but one of the ATPases are enhanced at high Ca2+, high pH, or high Na+, and that three ATPases are only expressed under stress conditions. Genes encoding ENA- or Na+,K+-ATPases were not found in the complete genomes of Arabidopsis thaliana and rice (Oryza sativa). On the basis of these results, we discuss the probable absence of Na+-ATPases in plants, and the function of Ca2+-ATPases that are expressed only under conditions of stress.

Molecular cloning and functional expression in bacteria of the potassium transporters CnHAK1 and CnHAK2 of the seagrass Cymodocea nodosa.

The cDNAs CnHAK1 and CnHAK2, encoding K+ transporters, were amplified from the leaves of the seagrass Cymodocea nodosa. None of these transporters suppressed the K+ deficiency of a Saccharomyces cerevisiae mutant, but both suppressed the equivalent defect of an Escherichia coli mutant. Overexpression of the transporter CnHAK1, but not CnHAK2, mediated very rapid K+ or Rb+ influxes in the E. coli mutant. The concentration dependence of these influxes demonstrated that CnHAK1 is a low-affinity K+ transporter, which does not discriminate between K+ and Rb+. CnHAK1 expressed in E. coli worked in reverse when the external K+ concentrations were low, and we established the condition of a simple functional test of K+ loss for transporters of the Kup-HAK family. In comparison with its homologue barley transporter HvHAK2, CnHAK1 was insensitive to Na+.

Cloning of two SOS1 transporters from the seagrass Cymodocea nodosa. SOS1 transporters from Cymodocea and Arabidopsis mediate potassium uptake in bacteria.

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na+-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na+, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na+ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K+-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K+. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K+ and Rb+ transporter. CnSOS1B mediated a transient, extremely rapid K+ or Rb+ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K+ and Rb+ uptake characteristics in bacteria.