Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.     

Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome

An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [³⁵S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome. Received: 4 September 1996 / Accepted: 30 November 1996     

Truncated presequences of mitochondrial F1-ATPase β subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco

The mitochondrial F₁-ATPase subunit (ATPase-) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH₂-terminal extension. By sequencing the mature N. tabacum ATPase-, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3 deletions in the sequence coding for the 90 NH₂-terminal residues of ATPase-. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and -glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase- remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Improvements of the Biological Diatom Index (BDI): Description and efficiency of the new version (BDI-2006)

The Biological Diatom Index (BDI) is a standardized method routinely used in France for the surveillance of watercourse quality. This index is based on a list of 209 key species showing different pollution sensitivities. The pollution sensitivity, or “ecological profile”, is determined through the species presence probability values along a seven quality classes gradient. In a sample, the presence and abundance of species from this list are used to calculate the BDI score. Recently, water managers pointed out several weaknesses of this index: (a) the BDI fails to correctly assess water quality in acidobiontic and brackish conditions, and (b) pollution sensitivity and tolerance of several key species used to calculate the index need to be improved.This paper presents and discusses the new version of the BDI, called BDI-2006. 2802 samples were used to create this new index (samples collected on all French river types during the last thirty years), where 1063 diatom species were identified. For each sample, the community was described with species relative abundance, and relevant physico-chemical parameters were collected: pH, conductivity, dissolved oxygen, biological oxygen demand, ammonium, orthophosphates and nitrates.Eight hundreds and thirty eight key species were finally kept, including species typical of acidic and brackish waters, tropical taxa and abnormal forms of many widespread taxa (as evidence of toxicological impacts).The physico-chemical and the biological datasets were explored with classical analysis (Principal Component Analysis) and neural networks (Self-Organizing Maps), combined to draw the new key species ecological profiles. Comparing to the initial version of the BDI, several species profiles (Nitzschia paleacea for example) were clearly enhanced.This new version was adopted by the French standardisation authority (AFNOR) in October 2007.     

Characterization of tomato (Solanum lycopersicum L.) mutants affected in their flowering time and in the morphogenesis of their reproductive structure

The impact of the season on flowering time and the organization and morphogenesis of the reproductive structures are described in three tomato mutants: compound inflorescence (s), single flower truss (sft), and jointless (j), respectively, compared with their wild-type cultivars Ailsa Craig (AC), Platense (Pl), and Heinz (Hz). In all environmental conditions, the sft mutant flowered significantly later than its corresponding Pl cultivar while flowering time in j was only marginally, but consistently, delayed compared with Hz. The SFT gene and, to a lesser extent, the J gene thus appear to be constitutive flowering promoters. Flowering in s was delayed in winter but not in summer compared with the AC cultivar, suggesting the existence of an environmentally regulated pathway for the control of floral transition. The reproductive structure of tomato is a raceme-like inflorescence and genes regulating its morphogenesis may thus be divided into inflorescence and floral meristem identity genes as in Arabidopsis. The s mutant developed highly branched inflorescences bearing up to 200 flowers due to the conversion of floral meristems into inflorescence meristems. The S gene appears to be a floral meristem identity gene. Both sft and j mutants formed reproductive structures containing flowers and leaves and reverting to a vegetative sympodial growth. The SFT gene appears to regulate the identity of the inflorescence meristem of tomato and is also involved, along with the J gene, in the maintenance of this identity, preventing reversion to a vegetative identity. These results are discussed in relation to knowledge accumulated in Arabidopsis and to domestication processes.     

Activation of the plasma membrane H+-ATPase by acid stress: Antibodies as a tool to follow the phosphorylation status of the penultimate activating Thr

Tight regulation of the plasma membrane proton pump ATPase (H⁺-ATPase) is necessary for controlling the membrane potential that energizes secondary transporters. This regulation relies on the phosphorylation of the H⁺-ATPase penultimate residue, a theonine, and the subsequent binding of regulatory 14-3-3 proteins, which results in enzyme activation. Using phospho-specific antibodies directed against the phosphorylable Thr of either PMA2 (Plasma membrane H⁺-ATPase from N. plumbaginifolia) or PMA4, we showed that the kinetics and extent of phosphorylation differ between both isoforms according to the growth or environmental conditions like cold stress.¹ Here, we used phospho-specific antibodies to follow PMA2 Thr phosphorylation upon acidification of the cytosol by incubating N. tabacum BY2 cells with four different weak organic acids. Increased PMA2 phosphorylation was observed for three of them, thus highlighting the role of the H+-ATPase in cell pH homeostasis.Key words: H⁺-ATPase, regulation, phosphorylation, phospho-specific antibodies, pH homeostasis, cold stressThe H⁺-ATPase is a major enzyme of the plant plasma membrane. This P-type ATPase couples ATP hydrolysis with proton transport out of the cell and establishes pH and potential gradients across the plasma membrane, thereby activating secondary transporters. At the physiological level, this enzyme is implicated in diverse roles, such as cytosolic pH regulation, cell elongation or stomata aperture.^2,3 H⁺-ATPase consists of ten membrane spanning regions and four cytosolic domains, among which the auto-inhibitory C-terminal region. The activation mechanism of the enzyme is well known and involves phosphorylation of its penultimate residue, a threonine, by an as yet unidentified protein kinase; phosphorylation in turn leads to the binding of regulatory 14-3-3 protein dimers and to the formation of an activated complex consisting of six H⁺-ATPases and six 14-3-3 proteins.^4–7Additional conserved phosphorylation sites in the enzyme C-terminal region have been shown to positively or negatively contribute to the enzyme regulation.^8–10 More sites have been discovered by large-scale phospho-proteomics, but have not been studied to date.^10,11 Most of these additional phosphorylated residues are located in the enzyme C-terminal autoinhibitory domain. This domain contains two to three inhibitory regions and a 14-3-3 binding region, partially super-imposed with an inhibitory region.^12,13 All these recent data suggest that the activity of H⁺-ATPase is finely tuned. However, the complexity of this regulation makes it difficult at the present stage to propose a comprehensive view.To follow and compare the activation status of two H⁺-ATPase isoforms belonging to different subfamilies, antibodies were designed for specifically recognizing the phosphorylated form of the penultimate Thr of either PMA2 (Plasma membrane H⁺-ATPase from N. plumbaginifolia) or PMA4, two broadly expressed isoforms belonging to subfamily I and II, respectively. This allowed us to find, for example, that PMA2, as opposed to PMA4, is strongly dephosphorylated upon cold stress. Both isoforms are strongly activated, upon subculturing N. tabacum BY2 suspension cells into a new media.¹ However, they underwent dephosphorylation at different rates as the cell culture proceeded. These data showed the usefulness of these antibodies for determining the regulation of specific H⁺-ATPase isoforms and better understanding their physiological roles.The primary function of the plasma membrane H⁺-ATPase is to transfer protons outside the cell. H⁺-ATPase is therefore considered as a possible regulator of the cytosolic pH homeostasis, for instance by preventing internal acidification. However, few data so far support this role for H⁺-ATPase. We addressed this point by adding to a N. tabacum BY2 cell culture weak organic acids, which are expected to permeate the membrane as a protonated form and dissociate once inside, resulting in cytosol acidification. This is expected to activate the plasma membrane H⁺-ATPase and so remove the proton excess out off the cell. A N. tabacum BY2 cell culture was treated with 5 mM of either citric acid, aminobenzoic acid, 2,2-dimethylglutaric acid, or propionic acid (Fig. 1). After several periods of time, a microsomal fraction was isolated and analyzed by western blotting. Among the four weak acids tested, propionic acid and citric acid induced strong and stable increase of PMA2 penultimate Thr phosphorylation. 2,2-dimethylglutaric acid induced a temporary increase of phosphorylation while aminobenzoic acid had no effect. The different responses might be explained either by different diffusion rates of the organic acids across the plasma membrane or by their possible toxicity. In addition, homeostasis of the intracellular pH results from the activity of several different enzymatic systems such as vacuolar H⁺-ATPase and H⁺-pyrophosphatase. Therefore it is also possible that, depending on the rate and/or extent of cytosol acidification, different responses are activated.Open in a separate windowFigure 1Effect of various weak acids on the phosphorylation of the PMA 2 penultimate Thr residue of N. tabacum suspension cells. A 3-day old N. tabacum BY2 cell culture was treated with 1/10^th volume of 50 mM of either aminobenzoic acid, 2,2-dimethylglutaric acid, citric acid or propionic acid, dissolved in the culture medium and brought beforehand to the same pH as the culture. After the indicated periods of time, cells were collected and a microsomal fraction was isolated and analyzed by western blotting using antibodies pThr⁹⁵⁵PMA 2 recognizing the PMA 2 penultimate activating Thr¹ (upper) and pan PMA 2 recognizing a short sequence specific for PMA2,¹⁴ (lower). C, untreated cells.This data supports the role H⁺-ATPase in pH homeostasis and highlights the strong potential of using phospho-specific antibodies to follow enzyme activation in the plant according to different environmental conditions. In addition, one should also take advantage of them as a tool for in vitro phosphorylation tests using different subcellular fractions of N. tabacum BY2 cells. This approach might lead to the isolation of the kinase and phosphatase involved in the modification of the penultimate Thr residue. Indeed, these enzymes are still undiscovered in spite of the fact that phosphorylation of this residue has been demonstrated more than a decade ago.