Article

Molecular Cloning and Sequence of cDNA Encoding the Plasma Membrane Proton Pump (H+-ATPase) of Arabidopsis thaliana

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Abstract

In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H+-ATPase) that produces an electric potential and pH gradient. We have isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H+-ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plot contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops.

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... Second, each plant has multiple PM H + -ATPase genes (11 in Arabidopsis) and each cell type can express several different isoforms. For example, wild-type Arabidopsis plants carrying knockouts of either of the two major PM H + -ATPase genes, Autoinhibited H + -ATPase isoform 1 and 2 (AHA1 and AHA2, respectively) (Harper et al., 1989;Pardo and Serrano, 1989;Baxter et al., 2003), grow normally under standard conditions, whereas the double knockout is embryo lethal (Haruta et al., 2010). Although these findings confirm that PM H + -ATPase is essential for plant growth, its specific physiological roles are unclear, and often only become apparent under stress conditions that reduce the transmembrane electrical gradient and/or external proton chemical gradient (Haruta et al., 2010;Haruta and Sussman, 2012). ...
... In Arabidopsis, AHA2 is the major root PM H + -ATPase (Harper et al., 1989), and the roots of aha2 mutants exhibit a reduced ability to acidify their surroundings (Haruta et al., 2010). Although the growth of aha2 plants is reduced in stress conditions that reduce the plasma membrane proton motive force, these plants are more resistant to toxic cations, such as lithium and cesium, than are wild-type plants (Haruta et al., 2010;Haruta and Sussman, 2012). ...
Article
The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the plasma membrane (PM), which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and, on this basis, it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals that regulate plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase.
... But Arabidopsis rapidly took over as the key species in which transporter genes have been first identified. The proton ATPase protein was identified from oat and subsequently cloned simultaneously from Nicotiana plumbaginifolia and Arabidopsis (Schaller & Sussman, 1988;Boutry et al., 1989;Harper et al., 1989;Pardo & Serrano, 1989). At the same time, Sauer & Tanner (1989) identified the first sugar transporters from Chlorella. ...
Article
The year 2014 marked the 25th International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.
... The first isolated plant transporter cDNAs were a phosphate translocator from spinach (Spinacia oleracea) chloroplasts (Flü gge et al., 1989), a hexose transporter from Chlorella kessleri (Sauer and Tanner, 1989) followed by three proton ATPases (Boutry et al., 1989;Harper et al., 1989;Pardo and Serrano, 1989). Within 5 years, the use of heterologous expression in yeast and functional characterization in Xenopus laevis oocytes led to the identification of genes encoding a number of physiologically important plant transporters. ...
Article
Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.
... The expression of H + ATPase is crucial for signal transduction in response to external environmental stimuli (Hentzen et al. 1996). Multiple ATPase gene families are reportedly also expressed in Arabidopsis thaliana (Harper et al. 1989;Pardo and Serrano 1989;Houlne and Boutry 1994;Nakajima et al. 1995). Therefore, the differential expression of this gene could suggest that it plays a strong role in attenuating drought stress. ...
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This study was carried out to identify drought-responsive genes in a drought tolerant faba bean variety (Hassawi 2) using a suppressive subtraction hybridization approach (SSH). A total of 913 differentially expressed clones were sequenced from a differential cDNA library resulted in a total of 225 differentially expressed ESTs. The genes of mitochondrial and chloroplast origin were removed, and the remaining 137 EST sequences were submitted to the gene bank EST database (LIBEST_028448). A sequence analysis identified 35 potentially drought stress-related ESTs that regulate ion channels, kinases, and energy production and utilization and transcription factors. Quantitative PCR on Hassawi 2 genotype confirmed that more than 65% of selected drought-responsive genes were drought-related. Among these induced genes, the expression levels of eight highly up-regulated unigenes were further analyzed across 38 selected faba bean genotypes that differ in their drought tolerance levels. These unigenes included ribulose 1,5-bisphosphate carboxylase (rbcL) gene, non-LTR retroelement reverse related, probable cyclic nucleotide-gated ion channel, polyubiquitin, potassium channel, calcium-dependent protein kinase and putative respiratory burst oxidase-like protein C and a novel unigene. The expression patterns of these unigenes were variable across 38 genotypes however, it was found very high in tolerant genotype. The up-regulation of these unigenes in majority of tolerant genotypes suggests their possible role in drought tolerance. The identification of possible drought responsive candidate genes in Vicia faba reported here is an important step towards the development of drought-tolerant genotypes that can cope with arid environments.
... Considerable progress has recently been made in the understanding of the molecular biology of plant PM H+- ATPases. Multiple genes encoding PM H+-ATPases have been cloned in tomato (Lycopersicon esculentum; Ewing et al., 1990; Ewing and Bennett, 1994 ), Nicotiana plumbaginifolia (Boutry et al., 1989; Perez et al., 1992; Moriau et al., 1993), Arabidopsis fhaliana (Harper et al., 1989Harper et al., , 1990Harper et al., , 1994; Pardo and Serrano, 1989; Houlne and Boutry, 1994), potato (Harms et al., 1994), rice (Wada et al., 1992; Ookura et al., 1994), and Vicia faba (L.E. Wimmers, unpublished data). ...
Article
The plant plasma membrane H+-ATPase energizes the secondary uptake of nutrients and may facilitate cell expansion by acidifying the cell wall. In yeast, Glc stimulates the accumulation of H+-ATPase mRNA, and the growth rate supported by various sugars is correlated with H+-ATPase protein abundance. Expression of three H+-ATPase genes, LHA1, LHA2, and LHA4, was previously detected in tomato (Lycopersicon esculentum). We have characterized the sequence of the LHA4 gene and examined the expression of these three tomato H+-ATPase genes in growing tissues and in response to exogenous sugars. LHA4 is a member of the H+-ATPase subfamily, including the Arabidopsis thaliana genes AHA1, AHA2, and AHA3. The 5[prime] untranslated region of the deduced LHA4 cDNA contains a short, open reading frame very similar to that in the Nicotiana plumbaginifolia gene PMA1. LHA4 transcript abundance in seedlings is correlated with cell growth, being 2.5 times greater in hypocotyls of dark- versus light-grown plants. The accumulation of both LHA4 and LHA2 mRNAs is induced by the addition of exogenous sugars and this induction appears to be dependent on sugar uptake and metabolism, because mannitol and 3–O-methylglucose do not stimulate mRNA acumulation. These results suggest that the induction of expression of H+-ATPase genes by metabolizable sugars may be part of a generalized cellular response to increased cell growth and metabolism promoted by the availability of an abundant carbon source.
... In the model plant Arabidopsis thaliana (Arabidopsis), 11 AUTOINHIBITED H + -ATPASE (AHA) genes encode for plasma membrane H + -ATPases (Axelsen and Palmgren 2001). AHA1 and AHA2 are the predominant plasma membrane H + -ATPases (Harper et al. 1989, Pardo and Serrano, 1989, Harper et al. 1990). AHA1 is a housekeeping protein found all over the plant, whereas AHA2 plays its major role in the roots. ...
Article
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Plasma membrane H⁺‐ATPase pumps build up the electrochemical H⁺ gradients that energize most other transport processes into and out of plant cells through channel proteins and secondary active carriers. In Arabidopsis thaliana, the AUTOINHIBITED PLASMA MEMBRANE H⁺‐ATPases AHA1, AHA2 and AHA7 are predominant in root epidermal cells. In contrast to other H⁺‐ATPases, we find that AHA7 is autoinhibited by a sequence present in the extracellular loop between transmembrane segments 7 and 8. Autoinhibition of pump activity was regulated by extracellular pH, suggesting negative feedback regulation of AHA7 during establishment of an H⁺ gradient. Due to genetic redundancy, it has proven difficult to test the role of AHA2 and AHA7, and mutant phenotypes have previously only been observed under nutrient stress conditions. Here we investigated root and root hair growth under normal conditions in single and double mutants of AHA2 and AHA7. We find that AHA2 drives root cell expansion during growth but that, unexpectedly, restriction of root hair elongation is dependent on AHA2 and AHA7, with each having different roles in this process. This article is protected by copyright. All rights reserved.
... With the advent of techniques of molecular biology, sequences of plasma membrane H + -ATPase genes were cloned from Arabidopsis thaliana (Harper et al., 1989(Harper et al., , 1990Serrano, 1989a, 1989b), Nicotiana plumbaginifolia (Boutry et al., 1989), and Lycopersicum esculentum (Ewing et al., 1990). This definitely showed the plant plasma membrane H + -ATPase not only to be evolutionarily related to its fungal counterparts but also to P-type ATPases of animals. ...
Article
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The plasma membrane H+‐ATPase of fungi and plants is a single polypeptide of fewer than 1,000 residues that extrudes protons from the cell against a large electric and concentration gradient. The minimalist structure of this nanomachine is in stark contrast to that of the large multi‐subunit FOF1 ATPase of mitochondria, which is also a proton pump, but under physiological conditions runs in the reverse direction to act as an ATP synthase. The plasma membrane H+‐ATPase is a P‐type ATPase, defined by having an obligatory phosphorylated reaction cycle intermediate, like cation pumps of animal membranes, and thus this pump has a completely different mechanism to that of FOF1 ATPases, which operates by rotary catalysis. The work that led to these insights in plasma membrane H+‐ATPases of fungi and plants has a long history, which is briefly summarized in this review.
... In fact, yeast is also a good system to investigate the plant plasma membrane proton ATPase. In plants, the formation of the proton gradient depends on a multigene family [219]. In Arabidopsis, there are three major isoforms (AHA1-3). ...
Article
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Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker’s yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein–protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.
... There are several mechanisms that could require this interaction. Ca 2+ bound to AGPs might be mobilized by transient extracellular acidification for example by local activation of plasma membrane H + -ATPases such as occurs in response to auxin (Harper et al., 1989;Fendrych et al., 2016 An additional or alternative role for binding Ca 2+ may be to contribute to the stability or function of certain AGPs at the cell-surface apoplast. It was described that a-dystroglycan, a highly glycosylated mammalian receptor essential for muscle and the nervous system function, utilizes a [GlcA-Xyl]-Ca 2+ interaction for binding with high affinity to laminin-a2 (Briggs et al., 2016). ...
Article
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Arabinogalactan proteins (AGPs) are a family of plant extracellular proteoglycans involved in many physiological events. AGPs are often anchored to the extracellular side of the plasma membrane and are highly glycosylated with arabinogalactan (AG) polysaccharides, but the molecular function of this glycosylation remains largely unknown. The beta-linked glucuronic acid (GlcA) residues in AG polysaccharides have been shown in vitro to bind to calcium in a pH-dependent manner. Here, we used Arabidopsis thaliana mutants in four AG beta-glucuronyltransferases (GlcAT14A, B, D and E) to understand the role of glucuronidation of AG. AG isolated from glcat14 triple mutants had a strong reduction in glucuronidation. AG from a glcat14a/b/d triple mutant had lower calcium binding capacity in vitro than AG from wild-type plants. Some mutants had multiple developmental defects such as reduced trichome branching. glcat14a/b/e triple mutant plants had severely limited seedling growth and were sterile, and the propagation of calcium waves was perturbed in roots. Several of the developmental phenotypes were suppressed by increasing the calcium concentration in the growth medium. Our results show that AG glucuronidation is crucial for multiple developmental processes in plants and suggest that a function of AGPs might be to bind and release cell-surface apoplastic calcium. m.
... Next, we tested our assumption that, in addition to pH cyt , repolarization kinetics and amplitude depend on the degree of depolarization. Although the first primary structure of a plant PM H + -ATPase and second crystal structures of AHA2 have been known since 1989 and 2007, respectively (33)(34)(35)(36), the H + pump protein has not yet been characterized electrophysiologically in AHA-free heterologous expression systems (37). To study the voltage-dependent properties of an Arabidopsis H + pump, in a system unaffected by regulation of the plant cell, we expressed WT AHA2 protein in Xenopus oocytes. ...
Article
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In plants, environmental stressors trigger plasma membrane depolarizations. Being electrically interconnected via plasmodesmata, proper functional dissection of electrical signaling by electrophysiology is basically impossible. The green alga Chlamydomonas reinhardtii evolved blue light-excited channelrhodopsins (ChR1, 2) to navigate. When expressed in excitable nerve and muscle cells, ChRs can be used to control the membrane potential via illumination. In Arabidopsis plants, we used the algal ChR2-light switches as tools to stimulate plasmodesmata-interconnected photosynthetic cell networks by blue light and monitor the subsequent plasma membrane electrical responses. Blue-dependent stimulations of ChR2 expressing mesophyll cells, resting around −160 to −180 mV, reproducibly depolarized the membrane potential by 95 mV on average. Following excitation, mesophyll cells recovered their prestimulus potential not without transiently passing a hyperpolarization state. By combining optogenetics with voltage-sensing microelectrodes, we demonstrate that plant plasma membrane AHA-type H ⁺ -ATPase governs the gross repolarization process. AHA2 protein biochemistry and functional expression analysis in Xenopus oocytes indicates that the capacity of this H ⁺ pump to recharge the membrane potential is rooted in its voltage- and pH-dependent functional anatomy. Thus, ChR2 optogenetics appears well suited to noninvasively expose plant cells to signal specific depolarization signatures. From the responses we learn about the molecular processes, plants employ to channel stress-associated membrane excitations into physiological responses.
... Different letters above the bars indicate significant differences at P < 0.05. It has been proposed that the negatively charged polar head groups may accelerate H þ pumping, which is consistent with the fact that PS is predominantly located on the cytoplasmic side where the catalytic site of the enzyme is found (Harper et al. 1989;Morales-Cedillo et al. 2015). Thus, the higher PS content in S. europaea and the transgenic Arabidopsis plants may help maintain K þ /Na þ homeostasis via maintaining PM potential under salinity (Fig. 10). ...
Article
Full-text available
Salinity-induced lipid alterations have been reported in many plant species, however, how lipid biosynthesis and metabolism are regulated and how lipids work in plant salt tolerance are much less studied. Here a constitutively much higher phosphatidylserine (PS) content in plasma membrane (PM) was found in the euhalophyte Salicornia europaea than Arabidopsis. A gene encoding phosphatidylserine synthase (PSS) was subsequently isolated from S. europaea, named SePSS, which was induced by salinity. Multiple alignments and phylogenetic analysis suggested SePSS belong to base-exchange-type PSS, which locates in endoplasmic reticulum. Knockdown of SePSS in S. europaea suspension cells resulted in reduced PS content, decreased cell survival rate, increased PM depolarization and K+ efflux under 400 or 800 mM NaCl. By contrast, upregulation of SePSS leads to increased PS and phosphatidylethanolamine (PE) levels and enhanced salt tolerance in Arabidopsis, along with lower accumulation of reactive oxygen species, less membrane injury, less PM depolarization and higher K+/Na+ in the transgenic lines than WT. These results suggest the positive correlation between PS levels and plant salt tolerance, and SePSS participates in plant salt tolerance by regulating PS levels, hence PM potential and permeability, which help maintain ion homeostasis. Our work provides a potential strategy for improving plant growth under multiple stresses.
... But Arabidopsis rapidly took over as the key species in which transporter genes have been first identified. The proton ATPase protein was identified from oat and subsequently cloned simultaneously from Nicotiana plumbaginifolia and Arabidopsis (Schaller & Sussman, 1988;Boutry et al., 1989;Harper et al., 1989;Pardo & Serrano, 1989). At the same time, Sauer & Tanner (1989) identified the first sugar transporters from Chlorella. ...
Article
I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. Natural variation and genome-wide association studies XIV. XV. XVI. XVII. References SUMMARY: The year 2014 marked the 25(th) International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.
... In recent years the identification of plant genes encoding P-type H+-ATPases has provided new molecular tools for their study. Initially, three genes cloned from Arabidopsis (isoforms AHA1, AHA2, and AHA3) were proposed to encode plasma membrane P-type H+-ATPases based on protein sequence similarities with (a) the funga1 H+-ATPases and (b) peptide sequences obtained from a 100-kD ATPase protein isolated from oat root plasma membranes (Harper et al., 1989;Pardo and Serrano, 1989). Gene families encoding similar proteins have been found in other plants such as tobacco (Boutry et ai., 1989) and tomato (Ewing and Bennett, 1994). ...
Article
More than 11 different P-type H+-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H+-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1–193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and [beta]-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H+-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H+-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.
... The kinetic results suggested a potential MPK6 regulation over the H + -ATPase activity through the differential expression of isoforms during cold acclimation. Arabidopsis thaliana expresses 11 different isoforms of the plasma membrane H + -ATPase, which are preferentially expressed in specific organs or tissues [63]. AHA1 and AHA2 genes are abundantly expressed in all organs and tissues [64]. ...
Article
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Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.
... pombe all display extensive sequence homology, as well as similar hydrophobicity profiles, thought to indicate 8-1 0 transmembrane domains (Aaronson et al., 1988). Plasma-membrane ATPases from flowering plants also have up to 36% amino acid sequence homology with these fungal enzymes (Harper et al., 1989;Pardo & Serrano, 1989;Boutry et al., 1989). This ATPase is so conserved in structure and function that should it be shown to influence stress tolerances in one organism, there would be grounds to suspect that its action might also determine tolerance levels in diverse fungi and plants. ...
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The pma1.1 mutations of Saccharomyces cerevisiae and Schizosaccharomyces pombe decrease plasma-membrane ATPase activity. This study investigated how they affect different stress tolerances, and the extent and duration of the heat-shock response. pma1.1 mutants exhibited higher resistance to ethanol and osmotic stress, but lower tolerance to ultraviolet damage, as compared to wild-type cells. pma1.1 mutations also increased tolerance of the lethal temperature of 48°C in cells in which no heat-shock response had been induced. However, after induction of a heat-shock response and elevated thermotolerance by a 25-38°C upshift, then maintaining cells at 38°C for 40 min, pma1.1 lowered subsequent tolerances of much higher lethal temperatures. Analysis of pulse-labelled S. cerevisiae proteins revealed reduced heat-shock protein synthesis in the pma1.1 mutant after a 25-38°C heat shock. This may explain the greater increases in thermotolerance in wild-type as compared to pma1.1 cells after both were given identical 25-38°C shocks. With more severe treatment (25-42°C), heat-shock protein synthesis in wild-type cells, although initially high, was switched off more rapidly than in the pma1.1 mutant. These results indicate that plasma-membrane ATPase action exerts a major influence over several stress tolerances, as well as the extent and duration of heat-shock protein synthesis following induction of the heat-shock response.
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This paper reportsthe identification and functional expression of a gene that is involved in nitrate uptake in plants, a process essential for the assimilation of nitrate and the biological removal of nitrate from the soil solution. The CHL1 gene of Arabldopsis, which when mutated confers resistance to the herbicide chlorate and a decrease in nitrate uptake, was isolated and found to encode a protein with 12 putative membrane-spanning segments. Injection of CHL1 mRNA into Xenopus oocytes produces a nitrate- and pH-dependent membrane depolarization, inward current, and nitrate uptake. These data show that the CHL1 gene encodes an electrogenic nitrate transporter. CHL1 mRNA is found predominantly in roots and displays nitrate- and pH-dependent regulation.
Article
Both lower and higher plants have been shown to possess efficient transport systems for the uptake of sugars across the plasmalemma. Genes encoding transport proteins for both mono- and disaccharides have been cloned recently. The main cloning strategies — differential screening, complementation cloning in Saccharomyces cerevisiae, and heterologous screening — are briefly summarized. The relationship of plant sugar transporters to a superfamily of more than 50 uni-, sym-, and antiporters cloned so far is discussed.Various possibilities for heterologous expression (in Schizosaccharomyces pombe, Saccharomyces cerevisiae, Xenopus oocytes) of plant sugar transporters are described and compared. Eight D-glucose transporters (from yeast to Arabidopsis to man) only possess 7% identical amino acids. First site-directed mutations of the Chlorella HUP1 transporter indicate that at least transmembrane helices 5, 7 and 11 line the D-glucose specific path through the membrane. The genomic structures of two plant transporters are outlined; the glycosylation of transport proteins as well as their tissue specificity is discussed.
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During the last 3 years, genes for plasma membrane H+-ATPases from fungi, protozoa and plants have been isolated. Sequence similarities indicate that H+-ATPases constitute a separate group with the family of ATPases with phosphorylated intermediates. Yeast is a convenient model system to approach the physiology of H+-ATPases by recombinant DNA methodologies. A mutational analysis of yeast H+-ATPase has demonstrated that the enzyme is essential and rate-limiting for growth. Intracellular pH homeostasis is one of the crucial functions of H+-ATPase. In addition, there are indications for the direct energization of some essential transport system. The regulation of ATPase activity is probably mediated by an interaction between the active site and an inhibitory domain at the carboxyl-terminus.
Chapter
The plasma membrane (PM) H+-ATPases are trans-membrane proteins, specifically present in plants and fungi. They build and maintain a steep proton gradient across the cell membrane required for the transport of ions and metabolites. The generated proton gradient is important for cell expansion as well as controls various physiological processes like stomatal functioning. The PM H+-ATPase is encoded by a multigene family that show tissue and developmental stage-specific expression. It belongs to a class of P-type ATPases and hence share a similar general mechanism of action but differs in its regulatory mechanisms. It also plays a significant role in governing various stress responses. In this chapter, recent advances in the field including structure, transcriptional, and posttranscriptional regulation along with the function of PM H+-ATPase are discussed. The role of PM H+-ATPase in mediating environmental stress response is also the focus of this chapter.
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Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.
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Accessions representing a range of diversity in Brassica napus and their potential diploid progenitor species were analyzed using nuclear, chloroplast (ct), and mitochondrial (mt) RFLPs. Based on RFLPs of ctDNA and mtDNA, cytoplasmic genomes of the diploid species examined could be classified into four major types, represented by almost all B. rapa, Broccoletto (a specialized form of B. rapa), B. oleracea, and B. montana. Results from phylogenetic analyses of these data suggest that B. montana might be closely related to the prototype that gave rise to both cytoplasms of B. rapa and B. oleracea. The four major types of cytoplasms found in the diploid species were also observed in B. napus accessions. A majority of the cultivated B. napus examined contained a cytoplasm different from those of either B. rapa and B. oleracea. These B. napus accessions (designated M/N cytoplasm type) had the same chloroplast genome as that of B. montana and a unique mitochondrial genome that was somewhat intermediate between those of B. montana and B. rapa. These results strongly support the concept of multiple origins of B. napus and provide the first evidence that most cultivated forms of B. napus were derived from a cross in which a closely related ancestral species of B. rapa and B. oleracea was the maternal donor. Phylogenetic relationships based on nuclear RFLPs clearly separated B. napus accessions having different cytoplasm types, providing further evidence for multiple origins of B. napus. Brassica napus accessions having M/N type cytoplasm were clustered together as three subgroups: one contained oilseed cultivars from Canada, a second contained oilseed cultivars from Europe, and a third contained accessions of rutabaga.
Article
In developing wheat grains (Triticum turgidum var. durum cv. Fransawi), post-sieve element transport of phloem-imported photoassimilates (sucrose) includes membrane transport, to and from the grain apoplasm, between symplasmically-isolated maternal and filial tissues. The cellular location and mechanism of these membrane transport steps were explored during rapid grain fill. Genomic Southern analysis indicated the presence of a multigene family of sucrose/H + symporters (SUTs). One or more SUTs were highly expressed in developing grains, as were P-type H + /ATPase(s) and a sucrose binding protein (SBP). Transcripts of these genes were detected in vascular parenchyma, nucellar projection and aleurone cells. Antibodies, raised against a SUT, an H + /ATPase and a SBP, were selectively bound to plasma membranes of vascular parenchyma cells, nucellar projection transfer cells and modified aleurone/sub-aleurone transfer cells. The nucellar projection transfer cells and modified aleurone/sub-aleurone transfer cells exhibited strong proton pumping activity. In contrast, SUT transport function was restricted to filial tissues containing modified aleurone/sub-aleurone transfer cells. Based on these findings, we conclude that SUTs expressed in maternal tissues do not function as sucrose/H + symporters. Membrane exchange from nucellar projection transfer cells to the endosperm cavity occurs by an as yet unresolved mechanism. Sucrose uptake from the endosperm cavity into filial tissues is mediated by a SUT localised to plasma membranes of the modified aleurone/sub-aleurone transfer cells.
Article
The plasma-membrane H(+)-ATPase (EC 3.6.1.35) of maize (Zea mays L.) coleoptiles and enclosed leaves has been localized at the optical-microscope level utilizing paraffin sections and a specific monoclonal antibody. Both in coleoptiles and in leaves the stomatal guard cells and the phloem of vascular bundles are the tissues most enriched in ATPase. The enrichment in guard cells is relevant to active ion transport during stomata opening. Considering the postulated activation of coleoptile ATPase by auxin, it is remarkable that the ATPase is not enriched in the coleoptile epidermis, where most of the auxin receptor is located.
Article
The auxin sensitivity of the plasma-membrane H(+)-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33-38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H(+)-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H(+)-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H(+)-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.
Article
Inorganic pyrophosphatase (PPase) was purified from membrane fractions isolated from Ricinus cotyledons. The non-ionic detergent dodecyl-ß-D-maltoside (lauryl maltoside) was used to solubilise the PPase from the phase-partitioned, upper phase fraction (plasma membrane-enriched), and purification was achieved using a combination of ion exchange chromatography and gel filtration. The PPase was resolved from the plasma membrane ATPase by exploiting the greater phospholipid dependency of elution of the PPase from a 300 SW gel filtration column. When the phospholipid concentration in the elution buffer was 0.5 mg/mL the PPase and ATPase eluted together but when it was lowered ten-fold the enzymes were resolved. The purification procedure resulted in an approximately 30-fold purification of the PPase from the original upper phase membranes with a yield of 20–25 %. The final purified fraction was enriched in a protein with an apparent molecular mass of 68 kDa which cross-reacted with an antibody raised to the mung bean tonoplast PPase. The purified PPase activity was markedly stimulated by potassium salts and inhibited by sodium fluoride, methylene diphosphonate, N,N′-dicyclohexylcarbodiimide and N-ethyl-maleimide with no significant inhibition by azide.
Article
Protein phosphorylation and regulation by phosphorylation of transport ATPases was studied in highly purified plasma membrane and tonoplast vesicles isolated from etiolated zucchini (Cucurbita pepo L.) hypocotyls. Plant lysophosphatidylcholine and the chemically similar animal lipid platelet-activating factor activated H+ transport activity of plasma membrane ATPase in vitro. In plasma membrane vesicles, purified by two-phase partitioning and subsequent free-flow electrophoresis, both lipids activated a membrane-associated protein kinase that phosphorylated a 97 kD polypeptide. A 97 kD polypeptide also gave a western blot signal generated by an antibody prepared against plasma membrane H+-ATPase. The phosphoprotein and the western blot signal were absent in tonoplast fractions purified by free-flow electrophoresis. In contrast, the B subunit of the tonoplast H+-ATPase was identified by western blot in those fractions but could not be found in plasma membrane fractions, corroborating membrane identity. When Ca2+-dependent and lysophospholipid-dependent protein phosphorylation in plasma membranes and in tonoplast were compared it was found for both membranes that the phosphorylation of several polypeptides was Ca2+-dependent but lysophospholipid-independent. These phosphoproteins were different in plasma membranes and tonoplast. Ca2+ is an established second messenger in plants, and lysophospholipids are proposed as potential signal mediators activating membrane-associated protein kinase(s), Our data indicate that they could be distinguished by their substrates. A function of both plasma membrane and tonoplast in plant signal transduction pathways is indicated.
Article
The localisation of H+-ATPase catabolites in subcellular fractions isolated from carnation petals has been determined immunologically using two antibodies, one raised against a mid-section of the protein (amino acids 340–650) and the other corresponding to the C-terminus. The native Mr 100000 polypeptide of the H+-ATPase was clearly discernible in Western blots of microsomal membranes probed with either antibody; each antibody also depicted a distinguishable pattern of lower abundance catabolites of the protein in microsomal membranes. Some of these catabolites were present in the cytosolic fraction as well, in higher abundance than in microsomal membranes. The antibodies also depicted distinguishable patterns of other catabolites of the H+-ATPase in the cytosol that were not present in microsomal membranes and that changed with advancing senescence of the petals. Immunocytochemical localization of the H+-ATPase and its catabolites using the antibody specific to the C-terminus showed staining of the cytoplasm as well as the plasma membrane. Moreover, the cytosolic H+-ATPase catabolites contain membrane-spanning domains of the protein and coeluted with phospholipid during Sepadex G-25 gel filtration. These observations indicate collectively that catabolites of the H+-ATPase formed within the plasma membrane are subsequently released into the cytosol, possibly in association with lipid.
Article
Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K⁺ uptake exist in plants. We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K⁺uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb⁺ uptake at micromolar concentrations with an apparent Km of ~22 μM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb⁺ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K⁺ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K⁺ nutrition and uptake into various plant cell types.
Chapter
Strongly hydrophobic integral membrane proteins are difficult to purify and characterise using conventional biochemical techniques. Eight years ago my co-workers and I initiated a research programme using molecular biological techniques to help elucidate the structure and function of important proteins in the plasma membrane of plant cells. We first turned out attention to the H+-ATPase, since this enzyme is easy to assay and is one of the more abundant transport proteins in this membrane (Surrowy and Sussman, 1987).
Article
The specificity of protein targeting processes is the basis of maintaining structural and functional integrity of the cell, enabling the various subcellular compartments to carry out their unique metabolic roles. Studies in plants have progressed markedly in the last 5 years, and many of the specific signals involved in the transport and targeting of proteins to the nucleus, chloroplast, mitochondrion and microbody, and to organelles along the secretory pathway (endoplasmic reticulum [ER], Golgi complex, and vacuole) have been characterized. Exciting prospects include the identification of receptors involved in the recognition of protein targeting signals, mechanisms of vesicle targeting, and the role of mRNA targeting. Although important exceptions exist, a striking feature of the mechanisms and cellular machinery of protein targeting is their universality — among plants, animals, and eukaryotic microorganisms — and even between prokaryotes and eukaryotes. More information is required about the structural features of proteins that allow for their stable accumulation in a particular subcellular compartment, of particular interest to the plant genetic engineer. Our understanding of the rules that govern protein folding and oligomer assembly and how these processes relate to a protein's ultimate stability in the cell is limited.
Chapter
The three major classes of membrane proteins are as follows: a. Peripheral or extrinsic proteins. These are associated with only one leaflet — either the outer or the inner — of the bilayer. b. Integral or intrinsic proteins. These are associated with both bilayer leaflets and may traverse the membrane more than once. c. Transport proteins. These may coincide with either ‘a’ or ‘b’ above and also include proteins which while interacting with the membrane, are not anchored in it and may traffic in the cytosol from one membrane type to the other. Transport proteins can be grouped into the following three general classes: 1. Pumps 2. Carriers 3. Channels
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Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H+ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H+ transport activity, namely, H+-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H+ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H+-ATPases mediate H+ extrusion, whereas most members of other six proteins mediate H+ influx, thus contributing to cytosolic pH homeostasis by directly modulating H+ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H+-coupled substrate influx, whereas other intra-family members facilitate H+-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H+-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H+ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H+ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.
Chapter
Hyperaccumulators are plants that can sequester heavy metals in their vacuoles and are capable of tolerating high amounts of such heavy metals. The metal tolerance mechanism is facilitated by overexpression of certain metal transport and binding proteins such as heavy metal transporters, phytochelatins, metallothioneins, etc. in various tissues. P-type ATPases constitute a large and diverse superfamily of transport proteins, which are of great importance in all organisms. They play a significant role in the maintenance of transition metal homeostasis, which, in turn, is extremely important for normal plant functioning. These proteins transport various metal ions such as Cu2 +, Cd2 +, Zn2 +, Ca2 +, H⁺, etc. across the cell membrane by a process characterized by hydrolysis of ATP during which a phosphorylated intermediate is formed. In addition to their role in metal hyperaccumulation, these proteins also help in the transport of metals to subcellular components and target proteins, metal detoxification, and micronutrient nutrition. This chapter describes the basic mechanism by which these proteins transport metals across the cell membrane, their classification of P-type ATPases based on the substrate(s) translocated by them, and the role of each type of P-type ATPase in maintaining metal homeostasis.
Article
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The plasma membrane of higher plants contains a H(+)-ATPase as its major ion pump. This enzyme belongs to the P-type family of cation-translocating enzymes and generates the proton-motive force that drives solute uptake across the plasma membrane. In Arabidopsis thaliana the plasma membrane H(+)-ATPase is encoded by a multigene family (Harper, J. F., Surowy, T. K., and Sussman, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1234-1238). The complete genomic sequence of a third Arabidopsis H(+)-ATPase isoform (referred to as AHA2) is presented here, and the predicted protein sequence is compared with previously published AHA1, AHA3, and tobacco Nicotiana plumbaginifolia NP1 isoforms. The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity. The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction. The 5' region contains a 23-base pair (bp) polypyrimidine sequence and a short upstream reading frame. In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region. An unusually large intron for Arabidopsis (greater than 1000 bp) is present at the beginning of the coding sequence of both AHA2 and AHA3. In the 3'-untranslated sequence of AHA1 and AHA2 but not AHA3, there is a 65-bp region of 85% identity and a second shorter region of 16-bp identity harboring an unusual putative poly(A) addition signal (dTTTGAAGAAACAAGGC). Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue.
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Ebenso wie Tiere verfügen Pflanzen über die Fähigkeit elektrische Signale zu generieren. Dabei repräsentieren elektrische Signale – Membranpotentialänderungen an der Plasmamembran – die frühesten Antworten, welche an Pflanzenzellen im Zuge veränderter externer und intrinsischer Bedingungen beobachtet werden können. Stimuli wie Kälte, Hitze, Verwundung, Herbivorie und Pathogene, aber auch physiologische Prozesse, wie Wachstum und Bestäubung führen zur Änderung des Potentials der Plasmamembran pflanzlicher Zellen. Die meisten dieser Membranpotentialänderungen bestehen aus einer schnellen Depolarisation, gefolgt von einer Repolarisation des Membranpotentials, deren Kinetik, in Abhängigkeit des Stimulus hoch variabel sein kann. Das Wissen über die molekularen Grundlagen der Generierung und Weiterleitung elektrischer Signale in Pflanzen ist im Gegensatz zu Tieren nur wenig verstanden. Eine Ausnahme stellen ‚klassisch-erregbare‘ Pflanzen wie die Venusfliegenfalle oder die Mimose dar. In diesen Pflanzen führt ein Berührungsreiz zur Auslösung eines charakteristischen Aktionspotentials, welches in der Folge zu einer, auf differentiellen Turgoränderungen basierenden, nastischen Bewegung führt. In allen anderen Pflanzen ist die Kinetik der Membranpotentialänderungen sehr variabel, abhängig vom Stimulus und dem physiologischen Zustand der Zellen und – mit Ausnahme der Reaktion auf einen Kältestimulus – lediglich nach langen Latenzzeiten wiederholbar. Dieser Umstand verhindert eine systematische Analyse der molekularen Basis elektrischer Signale in den meisten Pflanzen. Ziel dieser Arbeit war es daher, auf der Basis des Channelrhodopsin-2 (ChR2) aus der Grünalge Chlamydomonas reinhardtii, welches bereits seit 2005 in der Neurobiologie genutzt wird, ein nicht-invasives Werkzeug zur funktionellen Analyse elektrischer Signale in Pflanzen zu etablieren. ChR2 ist ein Blaulicht-aktivierter Kationenkanal, der für seine Funktion all trans-Retinal als Cofaktor benötigt. Im Rahmen dieser Arbeit wurden verschiedene Varianten des ChR2, mit einem Schwerpunkt auf ChR2-C128T und vor allem ChR2-D156C, auch bekannt als ChR2-XXL eingesetzt. ChR2 konnte bereits durch M. Baumann im Rahmen ihrer Dissertation funktionell im transienten Expressionssystem Nicotiana benthamiana dargestellt werden. In der vorliegenden Arbeit wurde das System weiter ausgebaut und die besonders aussichtsreichen ChR2-Varianten nicht nur in N. benthamiana, sondern auch in stabilen Arabidopsis thaliana Linien funktionell charakterisiert. Dabei konnte mit dem ChR2-XXL ein geeignetes optogenetisches Werkzeug zur Untersuchung elektrischer Signale in Pflanzen identifiziert werden. ChR2-XXL bietet die Möglichkeit das Membranpotential durch kurze, 5 s Blaulichtpulse im Mittel um 95 mV zu depolarisieren und im Anschluss die Repolarisationsphase zu untersuchen. Blaulicht-induzierbare, ChR2-XXL-vermittelte Depolarisationen konnten, reproduzierbar und beliebig oft an den gleichen Zellen wiederholt ausgelöst werden. Dadurch ermöglicht ChR2-XXL die bisher nur unzureichend bekannten molekularen Komponenten der Repolarisation des Membranpotentials in Pflanzen zu erforschen. In tierischen Zellen generieren spannungsabhängige Natriumkanäle die Depolarisation, während spannungsabhängige Kaliumkanäle die Depolarisationskinetik bestimmen. Die im Vergleich zu tierischen Zellen veränderten Ionengradienten lassen vermuten, dass die pflanzliche Depolarisation im Wesentlichen durch Ca2+-abhängige Anionenkanäle vermittelt wird, die durch den Efflux von Cl- das Membranpotential depolarisieren. Für die Repolarisation wird zum einen die Beteiligung von auswärtsgleichrichtenden Kaliumkanälen postuliert. Zum anderen wird auch eine Beteiligung der Plasmamembran (PM) H+-ATPasen vermutet, welche gleichzeitig einen essentiellen Beitrag zur Generierung des Ruhepotentials leisten. In der vorliegenden Arbeit wurde es durch den Einsatz von ChR2-XXL möglich, beide potentiellen Komponenten der Repolarisationsphase, Kaliumkanäle und PM H+-ATPasen, erstmals durch eine nicht-invasive, Anionen-unabhängige Methode der Depolarisation zu untersuchen. Durch den Einsatz von Mutanten und Kaliumkanalinhibitoren konnte ein möglicher Beitrag des auswärtsgleichrichtenden Kaliumkanals Arabidopsis thaliana GUARD CELL OUTWARD RECTIFYING K+ CHANNEL (AtGORK) an der Repolarisationsphase in Arabidopsis Mesophyllzellen nahezu ausgeschlossen werden. Der auswärtsgleichrichtende Kaliumkanal GORK öffnet erst bei Membranpotentialen positiv vom Gleichgewichtspotential für Kaliumionen (EK (-118 mV)). Da die ChR2-induzierbaren Depolarisationen ebenso wie viele natürliche Stimuli, diesen Wert kaum erreichen oder nur geringfügig überschreiten, leistet der GORK einen geringfügigen Beitrag bei der Repolarisation. Dies ließ vermuten, dass die Repolarisation von EK bis zum Ruhepotential bei ca. -180 mV dagegen möglicherweise durch die PM H+-ATPasen bewerkstelligt wird. Die Wirkung des PM H+-ATPase Inhibitors Natriumorthovanadat, sowie des PM H+-ATPase Aktivators Fusicoccin auf die Repolarisationsphase konnten diese Hypothese unterstützen. Die Hemmung der PM H+-ATPasen verlangsamte die Repolarisationskinetik während eine Aktivierung der PM H+-ATPasen diese beschleunigte. So wurde es erstmals möglich den genauen Einfluss der PM H+-ATPasen auf Wiederherstellung des Membranpotentials während der Repolarisation in Mesophyllzellen zu studieren. Darüber hinaus wurde beobachtet, dass in Gegenwart des Kaliumkanalblockers Ba2+ die Repolarisation ebenfalls beschleunigt werden konnte. In Übereinstimmung mit dem ‚Pump-and-Leak‘-Modell (Alberts et al. 2002) deutet dies darauf hin, dass schwach einwärtsgleichrichtende Kaliumkanäle, wie der ARABIDOPSIS K+ TRANSPORTER 2 (AKT2) dem PM H+-ATPasen Protonengradienten entgegenwirken und somit das Ruhepotential aus der Summe der bewegten Ladungen von Pumpen und Kaliumkanälen bestimmt wird. Das mögliche Potenzial optogenetischer, Rhodopsin-basierter Werkzeuge für die molekulare Analyse elektrischer Signale, insbesondere unter Einsatz der breiten Palette lichtgesteuerter Pumpen und Kanäle, ihrer spektralen Diversität und ihrer Einkreuzung in ausgewählte Arabidopsis Mutanten wird diskutiert.
Article
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The plasma membrane proton (H+)-ATPases of plants generate steep electrochemical gradients and activate osmotic solute uptake. H+-ATPase-mediated proton pumping orchestrates cellular homeostasis and is a prerequisite for plastic cell expansion and plant growth. All evidence suggests that the population of H+-ATPase proteins at the plasma membrane reflects a balance of their roles in exocytosis, endocytosis, and recycling. Auxin governs both traffic and activation of the plasma membrane H+-ATPase proteins already present at the membrane. As in other eukaryotes, in plants, SNARE-mediated membrane traffic influences the density of several proteins at the plasma membrane. Even so, H+-ATPase traffic, its relationship with SNAREs, and its regulation by auxin have remained enigmatic. Here, we identify the Arabidopsis (Arabidopsis thaliana) Qa-SNARE SYP132 (Syntaxin of Plants132) as a key factor in H+-ATPase traffic and demonstrate its association with endocytosis. SYP132 is a low-abundant, secretory SNARE that primarily localizes to the plasma membrane. We find that SYP132 expression is tightly regulated by auxin and that augmented SYP132 expression reduces the amount of H+-ATPase proteins at the plasma membrane. The physiological consequences of SYP132 overexpression include reduced apoplast acidification and suppressed vegetative growth. Thus, SYP132 plays unexpected and vital roles in auxin-regulated H+-ATPase traffic and associated functions at the plasma membrane.
Article
Drought stress is a limiting environmental factor that affects plant growth and development. The plant hormone abscisic acid (ABA) plays an important role in plant drought responses. Previous studies have indicated that ABA inhibits plasma membrane H+-ATPase (PM H+-ATPase) activity, and the decrease in PM H+-ATPase activity promotes stomatal closure under drought stress, thereby reducing water loss. However, the underlying molecular mechanisms are not well understood. In this study, we found that in Arabidopsis thaliana ABA induces an N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, namely, VESICLE-ASSOCIATED MEMBRANE PROTEIN 711 (VAMP711) to interact with the Arabidopsis PM H+-ATPases AHA1 and AHA2. The interaction occurs at their C-termini and inhibits PM H+-ATPase activity. Deletion of VAMP711 in Arabidopsis results in a higher PM H+-ATPase activity and slower stomatal closure in response to ABA and drought treatments. In addition, overexpression of VAMP711 partially rescues the drought-sensitive phenotype of ost2-2D, a mutation in AHA1 resulting in a constitutive activated PM H+-ATPase. Our results demonstrate that VAMP711 is involved in regulating ABA-mediated inhibition of PM H+-ATPase activity and stomatal closure in response to drought stress.
Chapter
The E1E2- or P-ATPases are a large and physiologically important family of cation pumps, widely distributed in both prokaryotic and eukaryotic cells (Table 1). In eubacteria, they play a series of specialized roles: scavenging potassium ions from the medium under conditions of K+ starvation, accumulating Mg2+, regulating the cytoplasmic Ca2+ and Cu2+ concentrations, and ridding the cell of toxic heavy metals such as Cd2+ and Zn2+. In fungi, as in other eukaryotic cells, the PATPases have taken on a more central function, creating the primary ion gradient that underlies virtually all nutrient uptake by an array of secondary, cation-coupled cotransporters. The fungal plasma-membrane H+-ATPase uses as much as a fourth of cellular ATP to extrude protons electrogenically (Gradmann et al. 1978); the resulting gradient (largely a membrane potential) distributes energy to H+-dependent cotransporters for sugars (Seaston et al. 1973; Slayman and Slayman 1974), amino acids (Eddy and Nowacki 1971; Seaston et al. 1973; Sanders et al. 1983), and inorganic ions (Rodriguez-Navarro et al. 1986). Plant cells resemble fungi in relying upon a proton gradient formed by a plasma-membrane H+-ATPase, while animal cells use a sodium gradient, produced by the Na+,K+-ATPase, to regulate cell volume and drive Na+-dependent cotransport of ions and nutrients.
Chapter
This chapter reviews the basic principles of the techniques used for intracellular pH measurement in the main cell compartments—namely, cytoplasm and vacuole. Alongwith being both substrate and product in numerous metabolic reactions, protons fulfill the regulatory role of coordinating the activities of enzyme-catalyzed pathways, membrane transport, and other regulators. The protons connect cellular compartments and also play important roles in intercellular traffic. The sudden pH shifts may impose critical loads on the cells. The technical improvements that have been brought about since the previously published reviews are described in the chapter. The technique involves the extraction of cell sap and the measurement of its pH with a glass electrode. It is used for various types of plant materials. The principle of this technique is based on three requirements: the probe molecule is metabolically inert, only the uncharged form is membrane permeant, and the probe is not to change the pH of the respective compartment. The distribution of protons within a plant cell appears as a critical element of cell organization and function.
Chapter
As a response to iron (Fe) deficiency, subapical swelling with formation of transfer cells was observed in root tips of a tolerant chickpea cultivar. Levels of H+-ATPase mRNA in the root tips within apical 10 mm were not influenced by the Fe supply to the medium. The findings indicate that the activity of H+-ATPase under Fe deficiency might be regulated at the post-transcriptional levels.
Chapter
The yeast plasma membrane [H+]-ATPase is essential for cell viability, pumping protons across the cell membrane to generate a large electrochemical gradient that provides the energy for nutrient uptake (reviewed by Goffeau amp; Slayman, 1981; Serrano, 1988; and Nakamoto amp; Slayman, 1989). The [H+]-ATPase is encoded by the PMA1 gene (Serrano et al., 1986) and belongs to a widely distributed family of transporters known as E1E2- or P-ATPases. Like other members of the group, it has a 100 kDa catalytic subunit that is firmly embedded in the lipid bilayer (Dufour amp; Goffeau, 1978) and alternates between two major conformational states (Ei and E2), hydrolyzing ATP by way of a covalent B-aspartyl phosphate reaction intermediate (Dame amp; Scarborough, 1981; Amory amp; Goffeau, 1982).
Chapter
Sorghum (Sorghum bicolor L. Moench) is one of the five most important grain crops worldwide, comprising the staple food source for over 300 million people in Africa and Asia (Doggett 1988). Sorghum is a very hardy plant that produces reasonable yields in locations with poor soils or limiting rainfall. In favorable environments, sorghum is a high yielding plant that has become a major feed crop throughout the developed and developing world. Sorghum is the closest relative to maize (Zea mays)and sugarcane (Saccharum species) among the major crops (Springer et al. 1989) and apparently arose as a unique species in Northeast Africa (Mann et al. 1983).
Chapter
Spatial and temporal oscillations occur in plants. Selected examples are used to show that macroscopic and rhytmic movements of plant organs arise form the activation and regulation of the electrogenic H+-ATPase and ion channels of the plasma membrane. In some cases, the electrocoupling between H+-ATPase and voltage-dependent ion channels could provide the basis for a membranar clock. Spatial patterns of pH and electrical potential along plants and plant cells could be a dissipative structure which arises when the H+-ATPase is working far from equilibrium.
Chapter
The alleviation of heavy metal stress affecting higher plants include the fields of uptake, translocation and efflux. In order to understand the arising problems first we should go through the present knowledge on these basic processes.
Article
The enzyme activity of the plasma membrane (PM) proton pump, well known as arabidopsis PM H⁺-ATPase (AHA) in the model plant arabidopsis (Arabidopsis thaliana), is controlled by phosphorylation. Three different classes of phytohormones, brassinosteroids (BRs), abscisic acid (ABA), and auxin regulate plant growth and responses to environmental stimuli, at least in part by modulating the activity of the pump through phosphorylation of the penultimate Thr residue in its carboxyl terminus. Here, we review the current knowledge regarding this tripartite hormonal AHA regulation and highlight mechanisms of activation and deactivation, as well as the significance of hormonal crosstalk. Understanding the complexity of PM H⁺-ATPase regulation in plants might provide new strategies for sustainable agriculture.
Article
Globally, micronutrient malnutrition has become a major health problem affecting over three billion people. Of the various micronutrients, problems (anemia, mental retardation, stunted growth, decreased immune function, and increased mortality) resulting from iron and zinc deficiencies are most prevalent and devastating in the developing countries. Rice serves as a staple food for more than half of the world population, but it has insufficient levels of the key micronutrients (Fe and Zn) to meet daily dietary requirements. Biofortification, which refers to the breeding of plants/crops with high bioavailable micronutrient content using conventional breeding, genetic engineering, and molecular and genomic approaches, has the potential to provide coverage for remote rural populations, where supplementation and fortification programs may not reach, and it inherently targets the poor who consume high levels of staple food and little else. Biofortified rice can be an effective solution to combat micronutrient malnutrition in developing countries with limited resources. The facts that substantial genetic variation for Fe and Zn contents exists in rice and that traits for high nutrient content can be combined with superior agronomic characteristics and high yield have allowed many scientists to use conventional breeding approaches to develop micronutrient-rich rice genotypes. Alternatively, genomic, transformation, and molecular tools have been used to improve our understanding of factors regulating micronutrient contents/bioavailability and rapid discovery of genes involved in iron uptake and storage in target tissues and consequently to develop novel high-Fe and/or high-Zn transgenic plants in rice. At CCS Haryana Agricultural University, Hisar, we have assessed variability for iron and zinc in a collection of 220 rice genotypes and identified several iron- and zinc-rich genotypes which have been used subsequently to raise mapping population and used for identification of QTLs for minerals in brown rice. Material is being used to select mineral-rich high-yielding rice genotypes.
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In order to identify Ca²⁺ ligands in the putative transmembrane domain 6 of the plasma membrane Ca²⁺ pump, amino acids Asn⁸⁷⁹, Met⁸⁸², Asp⁸⁸³, and Ser⁸⁸⁷ were singly altered. Asn⁸⁷⁹, Met⁸⁸², and Asp⁸⁸³ were chosen because the corresponding amino acids have been proposed as Ca²⁺ ligands in the sarcoplasmic reticulum Ca²⁺ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H.(1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca²⁺ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca²⁺ uptake and formed no acylphosphate from [γ-³²P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca²⁺ pump, these two are Ca²⁺ ligands. However, all the mutants at the position of Met⁸⁸² showed some activity. Indeed, the Met⁸⁸² → Ile mutant was fully active at a saturating Ca²⁺ concentration and only the K1/2 for Ca²⁺ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca²⁺ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca²⁺ pumps. The mutant Ser⁸⁸⁷ → Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca²⁺ ligand and could not replace the missing Thr at position Met⁸⁸²
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The plasma membrane of Neurospora crassa contains an electrogenic H+-ATPase (EC 3.6.1.35), for which we have isolated and sequenced both genomic and cDNA clones. The ATPase gene is interrupted by four small introns (58-124 base pairs). It encodes a protein of 920 amino acids (Mr, 99,886) possessing as many as eight transmembrane segments. The Neurospora ATPase shows significant amino acid sequence homology with the Na+,K+- and Ca2+-transporting ATPases of animal cells, particularly in regions that appear to be involved in ATP binding and hydrolysis.
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The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.
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The gastric H,K-ATPase is an active transport protein that is responsible for the maintenance of a large pH gradient across the secretory canaliculus of the mammalian parietal cell. Acid secretion across these epithelial cell membranes is coupled to the potassium-stimulated hydrolysis of ATP catalyzed by H,K-ATPase, but the mechanism of coupling between ion transport and ATP hydrolysis is unknown. In order to investigate the enzymatic mechanism of this coupling, a peptide derived from the ATP binding site of H,K-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate (FITC), into the active site before trypsin digestion of the protein. The labeling of the enzyme by FITC was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. At 100% inhibition of activity, 3.5 +/- 1.6 nmol of FITC were incorporated per mg of protein. The amino acid sequence of the active site peptide is His-Val-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Gln-Leu-Ser-Ile-Arg, and FITC reacts with the lysine. This sequence is very similar to sequences of fluorescein-labeled peptides from the ATP binding sites of Na,K-ATPase and Ca2+-ATPase, and suggests that the active site structures of these ion transport ATPases are similar.
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We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.
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Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation. The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase. A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated. For most of the experiments, the preparations were phosphorylated from [gamma-32P]ATP, denatured in acid, and subjected to proteolytic digestion. Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents. In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with [3H]borohydride. A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase. This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum. The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP. The corresponding sequence was different from that of the three ATPases. An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R. N. A. H., and McElhaney, R. N. (1983) Biochim. Biophys. Acta 735, 113-122).
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A synthetic DNA based on the known amino acid sequence of the brain/gut peptide cholecystokinin (CCK) was synthesized. This DNA contained deoxyinosines at ambiguous codon positions and was used as a probe to isolate the CCK gene directly from a human genomic library. Nucleotide sequence analysis of the isolated gene revealed that human preprocholecystokinin consists of 115 amino acid residues, with 11 amino acids in common with the human gastrin precursor, another member of the gastrin-CCK family, and that the coding region is separated by a single, long intron. CCK appears to be encoded by a single-copy gene in the haploid human genome, as revealed by genomic Southern hybridization analysis, suggesting that the same gene is expressed both in gut and brain.
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A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
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We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)(+) RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)(+) RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)(+) RNA hybridized with (32)P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.
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In crude extracts of plant tissue, the M(r) = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4 degrees C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases.
Article
A simple method for generating cDNA libraries from submicrogram quantities of mRNA is describe. It combines classical first-stand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161–170]. Neiher the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used. cDNA thus made can be tailed and cloned without further purification or sizing. Cloning efficiencies can be as high as 106 recombinants generated per μg mRNA, a considerable improvement over earlier methods. Using the fully sequenced1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.
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A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
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This chapter presents the basic principles of the use of scattered-light observation for macromolecular interaction kinetics. The pressure-jump technique has a particularly high potential for the study of macromolecular interactions because it is the molar volume of reaction that determines how well pressure drives a reaction. This technique is applicable to a very wide range of protein–protein interactions. One closely related technique has been described very recently in which repeated pressure pulses of small amplitude are used to force minor changes in the composition of the sample being monitored optically. In this apparatus, signal averaging over a very large number of pulses has been accomplished, thereby greatly increasing the signal-to-noise ratio. Additionally, pressure-jump studies using light scattered at 90 ° is explored in the chapter. This 90 ° light-scattering pressure-jump apparatus is capable of observing changes of only a few percent in the molecular composition of reacting systems having molecular weights as small as 48,000.
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The sensing of intrathoracic electrical impedance from an esophageal probe may allow relatively noninvasive monitoring of cardiac and respiratory functions of particular interest in anesthesia and intensive care. We have obtained a partial solution of the intrathoracic current-field problem for impedance measurements made from a four-terminal linear array of electrodes located in the esophagus. It allows prediction that aortic root motion will exceed aortic distension as a major determinant of the cardiac intrathoracic esophageal impedance signal. This prediction was confirmed for a specific carefully selected and placed electrode array in anesthetized dogs. In general, motions of organs will be more important than volume changes in affecting the esophageal impedance signal. Thus, timing information (preejection period and left ventricular ejection time) is available from electrodes on an esophageal probe, but cardiac output information appears to be inaccessible for fundamental reasons.
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When etiolated soybean seedlings are treated with the synthetic auxin, 2,4-dichlorophenoxy-acetic acid, cells of the mature hypocotyl become swollen and proliferate abnormally. This abnormal growth induced by auxin coincides with a 5- to 8-fold increase in the alpha-amanitin-insensitive RNA polymerase associated with isolated chromatin or nuclei. The alpha-amanitin-sensitive RNA polymerase activity of the auxin-treated hypocotyl was similar to that of control tissue. The increase in RNA polymerase I activity of chromatin and nuclei was maintained after solubilization and fractionation on DEAE-cellulose. Auxin thus appears to enhance RNA synthetic activity (i.e., ribosomal RNA) in mature soybean tissue by altering RNA polymerase I directly rather than by altering RNA polymerase I directly rather than by altering the chromatin template.
Article
Exposed portions of the 32 kd chloroplast membrane quinone-binding and triazine herbicide-binding protein of photosystem II have been mapped to the lumenal or to the outer (stromal) surface of the thylakoid by following reactions of antibodies generated against synthetic peptides corresponding to predicted hydrophilic amino acid sequences with normally oriented or everted membrane vesicles. These data have led to the construction of a model with five membrane-spanning domains. The model has been verified, in part, by immunoblots of fragments of the protein produced by trypsin treatment of thylakoids with peptide-specific antibodies. Some of the hydrophilic loops appear to be in close contact with proteins of the oxygen evolving complex of photosystem II inasmuch as their removal increases the antibody reaction.
Article
A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system; in fungi and plants an H+-pumping ATPase is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.
Article
Cation-pumping ATPases characterized by a phosphorylated intermediate have been proposed to contain kinase, phosphatase and transduction domains. Evidence is provided for this model by mutagenesis of critical residues in the proposed domains. The Glu233-Gln mutation blocks the turnover of the intermediate and serves to define the phosphatase domain. Mutations in aspartate residues 534, 560 and 638 alter the nucleotide specificity of the enzyme. These amino acids are therefore part of the ATP binding site. Lys474 seems to be essential for activity in this kinase domain. Finally, mutations in Asp378, the amino acid forming the phosphorylated intermediate, indicate that the formation of a phosphorylated intermediate is not an obligatory step in ATP hydrolysis but is required for coupling this process with proton pumping.
Article
We have cloned and sequenced complementary DNA encoding a Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum. We propose a model of the protein which has 3 cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow, penta-helical stalk. In this model, ATP bound to one cytoplasmic domain would phosphorylate an aspartate in an adjoining cytoplasmic domain, inducing translocation of Ca2+ from binding sites on the stalk.
Article
We have isolated and characterized a complementary DNA for the catalytic subunit of the sheep kidney sodium/potassium-dependent ATPase. The 1,016-amino-acid protein seems to have eight transmembrane domains. The apparent ouabain binding site is located at the extracellular junction of two transmembrane domains and is linked to the phosphorylation site by a 60-amino-acid conserved sequence that may be a major channel for energy transduction.
Article
The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.
Article
When the dog kidney Na+/K+-transporting ATPase (EC 3.6.1.37, formerly EC 3.6.1.3) was labeled with an ATP analogue, 5'-(p-fluorosulfonyl)benzoyladenosine (FSBA), there was a concomitant loss of ATPase activity. The presence of ATP protected the enzyme from both labeling and inactivation. The ATP-sensitive incorporation of FSBA is associated only with modification of the alpha subunit from which two labeled tryptic peptides were purified and sequenced. To establish any regions of the enzyme protruding from the membrane, the native Na+/K+-transporting ATPase from the electric ray, Torpedo californica, was treated with trypsin; and four peptides, which were released into the water phase, were purified and sequenced. A comparison of the peptide sequences with the deduced amino acid sequences of the DNA coding for the alpha subunit of T. californica and sheep kidney reveal the following. (i) FSBA-labeled peptides from the dog kidney enzyme are located in the central hydrophilic domain and show almost complete sequence homology with the same region in the alpha subunit from the electric ray and sheep kidney. Furthermore, the sequence homology of one of the two labeled peptides can be extended to the sarcoplasmic Ca2+-transporting ATPase and B subunit of Escherichia coli K+-transporting ATPase. (ii) Three trypsin-exposed peptides are found in the central hydrophilic domain, and one peptide is in the hydrophilic segment near the C terminus of the alpha subunit. (iii) The active center of Na+/K+-transporting ATPase is likely to be constructed from at least four different stretches in the primary sequence and, irrespective of the different specificity of cations, the various cation transport ATPases that form phosphorylated enzyme appear to have a common structure at the catalytic site for ATP hydrolysis.
Article
We have determined the DNA sequences of the genes encoding the three structural proteins of the Kdp-ATPase, an ATP-driven potassium transport system of Escherichia coli. Regions of the predicted amino acid sequence of KdpB, the phosphorylated protein of the system, are homologous to regions of the Ca2+-ATPase of rabbit sarcoplasmic reticulum. The phosphorylated aspartate residue of the latter is within a region of homology.
Article
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis and publication of biological sequence data. A group of programs that will interact with each research-article has been developed for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions for transfer are described.
Article
Syringomycin, a peptide toxin and a virulence factor produced by the bacterial phytopathogen Pseudomonas syringae pv. syringae, stimulated the phosphorylation of several plasma membrane polypeptides of red beet storage tissue. Among these was a 100-kDa polypeptide, which corresponds in size to the proton pump ATPase. The phosphorylations were insensitive to hydroxylamine, indicating that the polypeptide phosphorylated intermediates involved phosphate ester bonds characteristic of protein kinase-mediated phosphorylation. Phosphorylation of the 100-kDa polypeptide and of most of the other polypeptides was reduced or eliminated by extraction of the membranes with 0.1% (wt/vol) sodium deoxycholate, a treatment that also eliminated the ability of the toxin to stimulate ATPase activity. Phosphorylation of the 100-kDa polypeptide was highest with 10-20 mug of syringomycin; the same amounts gave the highest degree of ATPase activity stimulation. Phosphorylation of some of the polypeptides was eliminated or decreased by the Ca(2+) chelator EGTA. Addition of excess Ca(2+) restored the phosphorylation of most of these polypeptides. We conclude that syringomycin acts by stimulating an endogenous membrane protein kinase activity, which results in the phosphorylation of several membrane polypeptides. One of the phosphorylated polypeptides corresponds in size to the proton pump ATPase.
Article
In long-term experiments with differentially salinized nutrient solutions, plants of Lycopersicon esculentum Mill cv. Walter failed at Na(+) concentrations of 200 millimolar or more but tolerated K(+) concentrations of that magnitude. The behavior of the wild, salt-tolerant Lycopersicon cheesmanii (Hook) C. H. Mull., accession number 1401, was diametrically different; it tolerated Na(+) at 200 millimolar, but K(+) at the same concentration proved toxic to it.Short-term comparative studies on the absorption and translocation of Na(+), K(+), and Cl(-) of the two species were carried out using radioactive tracers with excised roots and whole plants. These studies showed that, under high salt conditions (50-100 millimolar NaCl), the tolerant 1401 freely accumulated Na(+) in the shoot, while the salt-sensitive cultivar excluded it from the leaves, where it has been shown to be toxic.In experiments where K(+) was limiting, the salt-tolerant species could partially substitute Na(+) for K(+). Sodium stimulated growth even when K(+) was present at adequate concentrations. The domestic cultivar could not substitute Na(+) for K(+) and showed no similar growth stimulation when Na(+) was added in the presence of adequate K(+). The salt-tolerant 1401 was more efficient in K(+) absorption than was the domestic cultivar at both low and moderate ambient K(+) concentrations.The two species differed little in their chloride relations.
Article
Arabidopsis thaliana is a small flowering plant with various properties that make it an excellent organism for experiments in molecular genetics. These properties include having a small nuclear genome, a near absence of dispersed repetitive DNA, and a generation time of 4 to 5 weeks. In addition, mutations that affect hormone synthesis and response, many different enzyme activities, and numerous developmental processes have been identified and characterized.
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