The H+-Translocating Inorganic Pyrophosphatase From Arabidopsis thaliana Is More Sensitive to Sodium Than Its Na+-Translocating Counterpart From Methanosarcina mazei.

Overexpression of membrane-bound K+-dependent H+-translocating inorganic pyrophosphatases (H+-PPases) from higher plants has been widely used to alleviate the sensitivity toward NaCl in these organisms, a strategy that had been previously tested in Saccharomyces cerevisiae. On the other hand, H+-PPases have been reported to functionally complement the yeast cytosolic soluble pyrophosphatase (IPP1). Here, the efficiency of the K+-dependent Na+-PPase from the archaeon Methanosarcina mazei (MVP) to functionally complement IPP1 has been compared to that of its H+-pumping counterpart from Arabidopsis thaliana (AVP1). Both membrane-bound integral PPases (mPPases) supported yeast growth equally well under normal conditions, however, cells expressing MVP grew significantly better than those expressing AVP1 under salt stress. The subcellular distribution of the heterologously-expressed mPPases was crucial in order to observe the phenotypes associated with the complementation. In vitro studies showed that the PPase activity of MVP was less sensitive to Na+ than that of AVP1. Consistently, when yeast cells expressing MVP were grown in the presence of NaCl only a marginal increase in their internal PPi levels was observed with respect to control cells. By contrast, yeast cells that expressed AVP1 had significantly higher levels of this metabolite under the same conditions. The H+-pumping activity of AVP1 was also markedly inhibited by Na+. Our results suggest that mPPases primarily act by hydrolysing the PPi generated in the cytosol when expressed in yeast, and that AVP1 is more susceptible to Na+ inhibition than MVP both in vivo and in vitro. Based on this experimental evidence, we propose Na+-PPases as biotechnological tools to generate salt-tolerant plants.

Vacuolar H(+)-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast.

Amine fungicides are widely used as crop protectants. Their success is believed to be related to their ability to inhibit postlanosterol sterol biosynthesis in fungi, in particular sterol-Δ(8),Δ(7)-isomerases and sterol-Δ(14)-reductases, with a concomitant accumulation of toxic abnormal sterols. However, their actual cellular effects and mechanisms of death induction are still poorly understood. Paradoxically, plants exhibit a natural resistance to amine fungicides although they have similar enzymes in postcicloartenol sterol biosynthesis that are also susceptible to fungicide inhibition. A major difference in vacuolar ion homeostasis between plants and fungi is the presence of a dual set of primary proton pumps in the former (V-ATPase and H(+)-pyrophosphatase), but only the V-ATPase in the latter. Abnormal sterols affect the proton-pumping capacity of V-ATPases in fungi and this has been proposed as a major determinant in fungicide action. Using Saccharomyces cerevisiae as a model fungus, we provide evidence that amine fungicide treatment induced cell death by apoptosis. Cell death was concomitant with impaired H(+)-pumping capacity in vacuole vesicles and dependent on vacuolar proteases. Also, the heterologous expression of the Arabidopsis thaliana main H(+)-pyrophosphatase (AVP1) at the fungal vacuolar membrane reduced apoptosis levels in yeast and increased resistance to amine fungicides. Consistently, A. thaliana avp1 mutant seedlings showed increased susceptibility to this amine fungicide, particularly at the level of root development. This is in agreement with AVP1 being nearly the sole H(+)-pyrophosphatase gene expressed at the root elongation zones. All in all, the present data suggest that H(+)-pyrophosphatases are major determinants of plant tolerance to amine fungicides.

Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer.

Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), together with other transporters, are responsible to a great extent for these symptoms. These multi-subunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across endocellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been shown to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target to control tumour cell proliferation. In the present review we present the major characteristics of this kind of proton pumps and we provide some recent insights on their in vivo regulation. Also, we review some of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and provide a brief summary of the studies related to cancer made recently with commercially available inhibitors. In the light of recent knowledge on the regulation of this proton pump, some new approaches to impair V-ATPase function are also suggested.

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast.

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

Intraorganellar acidification by V-ATPases: a target in cell proliferation and cancer therapy.

Vacuolar-type ATPases are multicomponent proton pumps involved in the acidification of single membrane intracellular compartments such as endosomes and lysosomes. They couple the hydrolysis of ATP to the translocation of one to two protons across the membrane. Acidification of the lumen of single membrane organelles is a necessary factor for the correct traffic of membranes and cargo to and from the different internal compartments of a cell. Also, V-ATPases are involved in regulation of pH at the cytosol and, possibly, extracellular milieu. The inhibition of V-ATPases has been shown to induce apoptosis and cell cycle arrest in tumour cells and, therefore, chemicals that behave as inhibitors of this kind of proton pumps have been proposed as putative treatment agents against cancer and many have been patented as such. The compounds filed in patents fall into five major types: plecomacrolides, benzolactone enamides, archazolids, chondropsins and indoles. All these have proved to be apoptosis inducers in cell culture and many to be able to reduce xenograft tumor growth in murine models. The present review will summarize their general structure, origin and mechanisms of action and put them in relation to the patents registered so far for the treatment of cancer.

N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast.

Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. In the present study, we show that the expression level and/or subcellular localization in S. cerevisiae of a heterologous type of multispanning membrane protein, the proton-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable N-terminal signal sequences. Chimaeric proteins were constructed by adding the putative N-terminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimaeric constructs not only substantially enhanced H+-PPase expression levels in transformed mutant cells, but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses demonstrated further the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana, a protein efficiently expressed in yeast in its natural form, alters the subcellular distribution of the chimaeras, suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP (green fluorescent protein)-H+-PPase fusion proteins.

H+-PPases: yesterday, today and tomorrow.

Suggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy-rich phosphate donor in biological energy conversion, were followed in the mid-1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membrane-bound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy-requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H(+)-PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H(+)-PPases differing in K(+) sensitivity were established. Over 200 H(+)-PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H(+)-PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high-molecular-weight polyphosphates (HMW-polyP). Here we will focus attention on the H(+)-PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures.

Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins.

Although several proton-pumping pyrophosphatases (H+-PPases) have been overexpressed in heterologous systems, purification of these recombinant integral membrane proteins in large amounts in order to study their structure-function relationships has proven to be a very difficult task. In this study we report a new method for large-scale production of pure and stable thermophilic H+-PPase from Thermotoga maritima. Following overexpression in yeast, a "Hot-Solve" procedure based on high-temperature solubilization and metal-affinity chromatography was used to obtain a highly purified detergent-solubilized TVP fraction with a yield around 1.5 mg of protein per litre of yeast culture. Electron microscopy showed the monodispersity of the purified protein and single particle analysis provided the first direct evidence of a dimeric structure for H+-PPases. We propose that the method developed could be useful for large-scale purification of other recombinant thermophilic membrane proteins.

Differential regulation of soluble and membrane-bound inorganic pyrophosphatases in the photosynthetic bacterium Rhodospirillum rubrum provides insights into pyrophosphate-based stress bioenergetics.

Soluble and membrane-bound inorganic pyrophosphatases (sPPase and H(+)-PPase, respectively) of the purple nonsulfur bacterium Rhodospirillum rubrum are differentially regulated by environmental growth conditions. Both proteins and their transcripts were found in cells of anaerobic phototrophic batch cultures along all growth phases, although they displayed different time patterns. However, in aerobic cells that grow in the dark, which exhibited the highest growth rates, Northern and Western blot analyses as well as activity assays demonstrated high sPPase levels but no H(+)-PPase. It is noteworthy that H(+)-PPase is highly expressed in aerobic cells under acute salt stress (1 M NaCl). H(+)-PPase was also present in anaerobic cells growing at reduced rates in the dark under either fermentative or anaerobic respiratory conditions. Since H(+)-PPase was detected not only under all anaerobic growth conditions but also under salt stress in aerobiosis, the corresponding gene is not invariably repressed by oxygen. Primer extension analyses showed that, under all anaerobic conditions tested, the R. rubrum H(+)-PPase gene utilizes two activator-dependent tandem promoters, one with an FNR-like sequence motif and the other with a RegA motif, whereas in aerobiosis under salt stress, the H(+)-PPase gene is transcribed from two further tandem promoters involving other transcription factors. These results demonstrate a tight transcriptional regulation of the H(+)-PPase gene, which appears to be induced in response to a variety of environmental conditions, all of which constrain cell energetics.

Proton-pumping inorganic pyrophosphatases in some archaea and other extremophilic prokaryotes.

Comparative studies between the proton-pumping, membrane-bound inorganic pyrophosphatases (H(+)-PPases) from hyperthermophilic and thermophilic prokaryotes and those from mesophilic organisms can now be performed because of very recent sequence data. Typical overall factors that contribute to protein thermostability are found in H(+)-PPases from extremophiles; nevertheless, putative active site motifs of this class of enzymes may be identical over the whole range of average growth temperatures of the compared prokaryotes. Heterologous expression in yeast of H(+)-PPases from organisms spanning a wide range of thermal habitats has allowed the biochemical comparison among these proteins within the same system, ensuring that differences observed are due to intrinsic characteristics of the proteins and not to their interactions with different cellular environments. On the other hand, the availability of H(+)-PPase sequences from a variety of sources have permitted molecular phylogenetic studies of this class of proton pumps, thus providing information about their general structural and functional properties. A great step forward may be expected when one of the several groups now attempting crystallization and 3D structural determination of H(+)-PPases will be successful.

Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases.

Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure-function relationships of this type of proton pumps.

Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa.

Proton-translocating inorganic pyrophosphatases (H(+)-PPase, EC 3.6.1.1) are integral membrane proteins that have been extensively studied in higher plants, the photosynthetic bacterium Rhodospirillum rubrum and, more recently, in some human pathogenic protozoa. By using a PCR-based approach, fragments of genes coding for H(+)-PPases in a number of protists, both free-living and parasites of animals and plants, that belong to diverse taxonomic groups (trypanosomatids, ciliates, apicomplexans, euglenoids, amoeboid mycetozoa, heterokonts) have been isolated. The experimental procedure involved the use of degenerate oligonucleotides designed from protein domains conserved in H(+)-PPases from plants and bacteria. The PCR-amplified DNA fragments exhibited the characteristic genomic structure and codon usage of the corresponding protozoan group. Paralogous genes were found in some species suggesting the occurrence of protein isoforms. These results indicate that H(+)-PPases are more widely distributed among protozoa than previously thought.