AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

A lithium-sensitive and sodium-tolerant 3'-phosphoadenosine-5'-phosphatase encoded by halA from the cyanobacterium Arthrospira platensis is closely related to its counterparts from yeasts and plants

3'-Phosphoadenosine-5'-phosphatase (PAPase) is required for the removal of toxic 3'-phosphoadenosine-5'-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+ for its activity and could use PAP or 3'-phosphoadenosine-5'-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50] = 3.6 mM) but only slightly sensitive to Na+ (IC50 = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.     

Cloning in Escherichia coli K-12 of a Na+-dependent transport system from a marine bacterium.

The transport of D-alanine by Escherichia coli K-12 neither requires nor is stimulated by Na+. The transport of D-alanine by the marine bacterium Alteromonas haloplanktis 214 requires Na+ specifically. Mutants of E. coli which were unable to transport D-alanine were isolated by enrichment for D-cycloserine resistance. One of the mutants was transformed with a gene bank of A. haloplanktis chromosomal DNA. Two transformants, E. coli RM1(pPM1) and E. coli RM1(pPM2) were able to transport D-alanine by a Na+-dependent mechanism. Li+ and K+ were unable to replace Na+. Both transformants contained chimeric plasmids with inserts which hybridized with A. haloplanktis but not E. coli chromosomal DNA or each other. Despite the lack of homology between the inserts, Na+-dependent D-alanine transport in the two transformants could not be distinguished either by kinetic studies or by differences in the capacity of various amino acids to compete for D-alanine uptake.     

The arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes.

In saline environments, plants accumulate Na(+) in vacuoles through the activity of tonoplast Na(+)/H(+) antiporters. The first gene for a putative plant vacuolar Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and shown to increase plant tolerance to NaCl. However, AtNHX1 mRNA was up-regulated by Na(+) or K(+) salts in plants and substituted for the homologous protein of yeast to restore tolerance to several toxic cations. To study the ion selectivity of the AtNHX1 protein, we have purified a histidine-tagged version of the protein from yeast microsomes by Ni(2+) affinity chromatography, reconstituted the protein into lipid vesicles, and measured cation-dependent H(+) exchange with the fluorescent pH indicator pyranine. The protein catalyzed Na(+) and K(+) transport with similar affinity in the presence of a pH gradient. Li(+) and Cs(+) ions were also transported with lower affinity. Ion exchange by AtNHX1 was inhibited 70% by the amiloride analog ethylisopropyl-amiloride. Our data indicate a role for intracellular antiporters in organelle pH control and osmoregulation.     

Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase

The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance.     

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.     

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.     

Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1 , a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli , is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.     

Characterization of the arabidopsis formin-like protein AFH1 and its interacting protein

A partial cDNA encoding an Arabidopsis thaliana FH (Formin Homology) protein (AFH1) was used as a probe to clone a full length AFH1 cDNA. The deduced protein encoded by the cDNA contains a FH1 domain rich in proline residues and a C-terminal FH2 domain which is highly conserved amongst FH proteins. In contrast to FH proteins of other organisms, the predicted AFH1 protein also contains a putative signal peptide and a transmembrane domain suggesting its association with membrane. Cell fractionation by differential centrifugation demonstrated the presence of AFH1 in the Triton X-100 insoluble microsomal fraction. An Arabidopsis cDNA library was screened to identify proteins that interact with the C-terminal region of AFH1 using yeast two-hybrid assays, and one of the isolated cDNAs encoded a novel protein, FIP2. Experiments using recombinant proteins expressed in E. coli demonstrated that FIP2 interacted directly with AFH1. The amino acid sequence of FIP2 has partial homology to bacterial putative membrane proteins and animal A-type K+ ATPases. AFH1 may form a membrane anchored complex with FIP2, which might be involved in the organization of the actin cytoskeleton.     

The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter.

The high-affinity K+ uptake system of plants plays a crucial role in nutrition and has been the subject of extensive kinetic studies. However, major components of this system remain to be identified. We isolated a cDNA from barley roots, HvHAK1, whose translated sequence shows homology to the Escherichia coli Kup and Schwanniomyces occidentalis HAK1 K+ transporters. HvHAK1 conferred high-affinity K+ uptake to a K(+)-uptake-deficient yeast mutant exhibiting the hallmark characteristics of the high-affinity K+ uptake described for barley roots. HvHAK1 also mediated low-affinity Na+ uptake. Another cDNA (HvHAK2) encoding a polypeptide 42% identical to HvHAK1 was also isolated. Analysis of several genomes of Triticeae indicates that HvHAK1 belongs to a multigene family. Translated sequences from bacterial DNAs and Arabidopsis, rice, and possibly human cDNAs show homology to the Kup-HAK1-HvHAK1 family of K+ transporters.     

Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana.

Both genomic and full length cDNA clones of an Arabidopsis thaliana sugar carrier, STP1, have been obtained using a cDNA clone of the H+/hexose cotransporter from the green alga Chlorella kessleri as hybridization probe. The peptide predicted from these sequences in 522 amino acids long and has a molecular weight of 57,518 kd. This higher plant sugar carrier contains 12 putative transmembrane segments and is highly homologous to the H+/hexose cotransporter from Chlorella, with an overall identity in the amino acid sequence of 47.1%. It is also homologous to the human HepG2 glucose transporter (28.4%), and other sugar carriers from man, rat, yeast and Escherichia coli. The definite proof for the function of the STP1 protein as a hexose transporter and data on its substrate specificity were obtained by heterologous expression in the fission yeast Schizosaccharomyces pombe. Transformed yeast cells transport D-glucose with a 100-fold lower KM value than control cells. Moreover only the transformed cells were able to accumulate the non-metabolizable D-glucose analogue 3-O-methyl-D-glucose, indicating that the Arabidopsis carrier catalyses an energy dependent, active uptake of hexoses. Expression of STP1 mRNA is low in heterotrophic tissues like roots or flowers. High levels of expression are found in leaves.     

Gene cloning and characterization of VcrM,a Na+-coupled multidrug efflux pump,from Vibrio cholerae non-O1

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments. A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump.     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail

Recently, a cyanobacterium Synechocystis sp. PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB). Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes. It turned out that A. halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity. Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E. coli mutant. The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH. The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E. coli mutant but not the Li(+)-sensitive phenotype. The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter. These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A. halophytica.     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

Molecular and functional analysis of a vacuolar Na+/H+ antiporter gene of Rosa hybrida

A vacuolar Na+/H+ antiporter gene was isolated from Rosa hybrida (RhNHX1). The amino acid sequence encoded by the RhNHX1 cDNA shows homology to that of the yeast NHX1. The cDNA contains 2080 nucleotides and an open reading frame of 1632 nucleotides that encodes a protein of 543 amino acids with a deduced molecular mass of 60,045 daltons. The deduced amino acid sequence of RhNHX1 is 74.1% identical to that of a vacuolar Na+/H+ antiporter of Arabidopsis thaliana, AtNHX1, and contains the consensus amiloride-binding domain. RhNHX1 suppressed the hygromycin-sensitive phenotype of the yeast nhx1 mutant. In addition, the expression of RhNHX1 in rose increased in the presence of NaCl. These results suggest that the product of RhNHX1 functions as a vacuolar Na+/H+ antiporter in rose plants.