Rosario Haro

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.

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.

HKT1 mediates sodium uniport in roots. Pitfalls in the expression of HKT1 in yeast.

The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K+-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na+-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K+-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.

Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae

Two Neurospora crassa genes, trk‐1 and hak‐1, encode K+ transporters that show sequence similarities to the TRK transporters described in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and to the HAK transporters described in Schwanniomyces occidentalis and barley. The N. crassa TRK1 and HAK1 transporters expressed by the corresponding cDNAs in a trk1Δ trk2Δ mutant of S. cerevisiae exhibited a high affinity for Rb+ and K+. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1Δ trk2Δ mutant with the kinetic characteristics of K+ uptake in N. crassa cells allowed TRK1 to be identified as the dominant K+ transporter and HAK1 as a transporter that is only expressed when the cells are K+ starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K+–H+ symporter described in N. crassa, whereas TRK1 might be a K+ uniporter. Although the co‐existence of K+ transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co‐existence may be the normal situation in soil fungi.

The twins K+ and Na+ in plants.

In the earths crust and in seawater, K(+) and Na(+) are by far the most available monovalent inorganic cations. Physico-chemically, K(+) and Na(+) are very similar, but K(+) is widely used by plants whereas Na(+) can easily reach toxic levels. Indeed, salinity is one of the major and growing threats to agricultural production. In this article, we outline the fundamental bases for the differences between Na(+) and K(+). We present the foundation of transporter selectivity and summarize findings on transporters of the HKT type, which are reported to transport Na(+) and/or Na(+) and K(+), and may play a central role in Na(+) utilization and detoxification in plants. Based on the structural differences in the hydration shells of K(+) and Na(+), and by comparison with sodium channels, we present an ad hoc mechanistic model that can account for ion permeation through HKTs.

Regulation of potassium fluxes in Saccharomyces cerevisiae

To investigate the regulation of K+ fluxes in Saccharomyces cerevisiae the dependence of K+ efflux and Rb+ influx on [K+]i, pHi, [Na+]i, membrane potential, cell volume, and turgor pressure were studied in cells with different K+ contents. By decreasing the cell volume with osmotic shocks and the cellular pH with butyric acid the following was found. (1) The K+ efflux induced by uncouplers decreases simultaneously with the decrease of the K+ content of the cell, but the process was insensitive to [K+]i, pHi, cell volume and turgor pressure. The internal presence of Na+ inhibited this K+ efflux. (2) The increase of the Vmax of Rb+ influx observed in low-K+ cells is due to the decrease of the pHi and probably mediated by the increase of the activity of the plasma membrane ATPase. The Vmax is independent of [K+]i, [Na+]i, cell volume and turgor pressure. (3) The decrease in the Km of Bt+ influx observed in low-K+ cells does not depend directly on [K+]i, pHi, cell volume or turgor pressure. If Na+ is present, [Na+]i might be directly involved in the regulation of the Km.

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na(+) ] and [K(+) ] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na(+) -selective class I-HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single-nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near-isogenic lines (NILs): 157-14 (double homozygote for the cheesmaniae alleles) and 157-17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157-14 in comparison with 157-17. A significant difference between NILs was also found for [K(+) ] and the [Na(+) ]/[K(+) ] ratio in leaf and stem but not for [Na(+) ] arising a disagreement with the corresponding RIL population. Their association with leaf [Na(+) ] and salt tolerance in tomato is also discussed.

Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae

The TRK-HKT family of K(+) transporters mediates K(+) and Na(+) uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K(+) or Rb(+), both of which bind the two binding sites of ScTRK1 with very high affinities in K(+)-starved cells. Na(+) can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K(+) and Na(+), or Rb(+) and Na(+), the transporter takes up Na(+) together with K(+) or Rb(+). Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln(90) is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu(949) is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K(+) uptake. This is not Na(+)-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K(+)-H(+) cotransport.

Phylogenetic Analysis of K+ Transporters in Bryophytes, Lycophytes, and Flowering Plants Indicates a Specialization of Vascular Plants

As heritage from early evolution, potassium (K+) is absolutely necessary for all living cells. It plays significant roles as stabilizer in metabolism and is important for enzyme activation, stabilization of protein synthesis, and neutralization of negative charges on cellular molecules as proteins and nucleic acids. Land plants even enlarged this spectrum of K+ utilization after having gone ashore, despite the fact that K+ is far less available in their new oligotrophic habitats than in sea water. Inevitably, plant cells had to improve and to develop unique transport systems for K+ accumulation and distribution. In the past two decades a manifold of K+ transporters from flowering plants has been identified at the molecular level. The recently published genome of the fern ally Selaginella moellendorffii now helps in providing a better understanding on the molecular changes involved in the colonization of land and the development of the vasculature and the seeds. In this article we present an inventory of K+ transporters of this lycophyte and pigeonhole them together with their relatives from the moss Physcomitrella patens, the monocotyledon Oryza sativa, and two dicotyledonous species, the herbaceous plant Arabidopsis thaliana, and the tree Populus trichocarpa. Interestingly, the transition of green plants from an aqueous to a dry environment coincides with a dramatic reduction in the diversity of voltage-gated potassium channels followed by a diversification on the basis of one surviving K+ channel class. The first appearance of K+ release (Kout) channels in S. moellendorffii that were shown in Arabidopsis to be involved in xylem loading and guard cell closure coincides with the specialization of vascular plants and may indicate an important adaptive step.

Functional analysis of the M2D helix of the TRK1 potassium transporter of Saccharomyces cerevisiae

Eukaryotic KcsA-related K+ transporters mediate physiologically relevant K+ and Na+ fluxes in fungi and plants. ScTRK1 is a characteristic member of the group, and here we report a mutational analysis of the unique M2(D) helix of this transporter. Our results support the theoretical models placing this helix in a relevant position in the pore and interacting with P segments. Most single mutations eliminating positively charged or introducing negatively charged residues reduced the V(max) of Rb+ influx to a half, several together showed an additive effect, and four practically suppressed transport. In contrast, the introduction of only one positively charged residue practically abolished the function of the transporter. Almost all mutations in the M2(D) helix affected the two Rb+ binding sites of the transporter, mimicking mutations in the selectivity filter.