K+-dependent enzyme activity of H+-ATPase in the E. coli membrane

It was shown that DCCD-sensitive ATPase activity of isolated membranes and preparations of F1F0 only from anaerobically grown E. coli depended on K+ activity. F1F0 include two additional proteins which correspond to the Trk system. The data improve the possibility to form supercomplex (F1F0-Trk) functioning as the H(+)-K(+)-pump.     

Formation of an ion transport supercomplex in Escherichia coli. An experimental model of direct transduction of energy

Hydrogen gas production was observed to occur during ATP-driven H+/K+ exchange in anaerobically grown E. coli. Neither process was found in aerobically grown cells or anaerobic cells grown on nitrate medium or when the osmotic pressure was decreased or K+ removed, or finally when DCCD, arsenate or CCCP was applied. Dithiothreitol restored the process even in the presence of CCCP but not in other cases of inhibition. A model of a multienzyme transport super-complex is proposed. The supercomplex consists of three genetically independent mechanisms: F0F1 H+-ATPase to provide energy, the K+-transporting Trk system as energy sink and formate-hydrogen lyase as donor of reducing equivalents. Within this supercomplex direct transduction of energy is accomplished via oxidation of 2 SH to S-S.     

N,N'-dicyclohexylcarbodiimide-sensitive K+-dependent H+ secretion from anaerobically grown E. coli cells

Dependence of N,N'-dicyclohexylcarbodiimide (DCC)-sensitive H+ secretion of K+ activity was discovered. This dependence took place only in anaerobically grown bacteria, and only at the structural intact DCC-sensitive H+-ATPase complex and K+-ionophore Trk.     

Interaction of H+ and K+ transport systems in E. coli growing under anaerobic and aerobic conditions

The interaction of H+-ATPase complex F1 X F0 with the Trk system of K+ accumulation in E. coli grown quasi-anaerobically in pepton media with glucose (anaerobia) and aerobically in the salt medium with succinate (aerobia) treated with cyanide was studied. The ratio of H+ fluxes via F1 X F0 and K+ fluxes via the Trk system is stable and equals 2 in anaerobia and is changed from 0.5 to 5.0 in aerobia treated with cyanide in response to pH variation, K+ activity and temperature variations. Q10 is about 2.8 both for F1 X F0 and the Trk system in anaerobia, but 2.4 and 1.0 respectively in aerobia. K+ distribution in anaerobia reaches high values, K+ equilibrium potential is much higher than the measured membrane potential. K+ distribution in aerobia is smaller, which is in conformity with the measured membrane potential. Structural association of F1 X F0 and the Trk system with the formation of H+--K+-pump is assumed to take place in anaerobia, and separate operation of these systems occurs in aerobia, transfer of K+ via Trk system being energized by the electric field on the membrane.     

The capacity of anaerobically grown bacteria to exchange the 2H+ of the cell for the K+ of the medium and to maintain a high K+ distribution between cell and medium

The N,N'-dicyclohexylcarbodiimide sensitive exchange of 2H+ of a cell for K+ of medium stable to pH, K+ activity and temperature changes has been discovered in anaerobically grown gram-negative Escherichia coli, Salmonella typhimurium. S. enteritidis, Proteus mirabilis, P. vulgaris, anaerobic gram-positive bacteria Streptococcus faecalis, Lactobacillus salivarius, L. lactis in the presence of exogenic energy source. This exchange in gram-negative bacteria is operating only at increase of medium osmolarity. The high K+ distribution between cell and medium has been reached during the exchange of 2H+ for one K+ and the corresponding potassium equilibrium potential is much more than the measured delta psi. In aerobically grown E. coli, S. typhimurium, Brevibacterium flavum and aerobic Micrococcus luteus exchange of 2H+ for K+ does not take place, the K+ distribution is lower and in good conformity with the measured delta psi. It is assumed that exchange of 2H+ for K+ in anaerobic bacteria is carried out by the H+-ATPase complex and the Trk (or Trk-like) system of K+ absorption united into the same membrane supercomplex which functions as the H+-K+-pump and supports the high K+ distribution between cell and medium.     

The nature of H+-K+-exchange in anaerobically and aerobically grown S. typhimurium

H+-K+-exchange via the Trk-like system of K+ accumulation takes place in anaerobically grown S. typhimurium LT-2 with stable ratio of DCC-sensitive ionic fluxes, equal to 2H+ of a cell for one K+ of the medium. This exchange is now observed in the mutant S. typhimurium TH-31 with unfunctional H+-ATPase. H+-K+-exchange in aerobically grown S. typhimurium LT-2 has unstable ratio of ionic fluxes. The rate of K+ uptake in anaerobically grown bacteria is higher than that in the aerobically grown ones. Q10 is about 1.8 both for H+ transfer and K+ uptake in anaerobically grown bacteria, but it is 1.7 and 0.9 respectively in the aerobically grown ones. Delta psi is not changed by different temperatures both in anaerobically and aerobically grown bacteria. The distribution of K+ in anaerobically grown bacteria is higher than 10(3) and the potassium equilibrium potential is much higher than the measured delta psi. In aerobically grown bacteria the distribution of K+ is in good conformity with the measured delta psi. H+ and K+ transport in anaerobically grown cells is likely to proceed by the same mechanism, which includes H+-ATPase and the Trk-like system. In aerobically grown bacteria these transport systems work separately, and the Trk-like system as K+-ionophore serving for K+ uptake across the electrical field on the membrane.     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F0F1 ATP-synthase and formate oxidation by formate hydrogen lyase (FHL) has been established in inverted membrane vesicles of Escherichia coli JW 136 mutant with double deletions (delta hya/ delta hyb) of hydrogenase 1 and 2 grown anaerobically on glucose in the absence of external electron acceptors (pH 6.5). ATP synthesis was suppressed by H+ -ATPase inhibitors N,N'-dicyclohexylcarbodiimide (DCCD) and sodium azide as well as by the protonophore carbonyl cyanide-m-chlorophenyhydrazone (CCCP). Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of vesicles. The maximal rate of ATP synthesis (0.83 microM/min x mg protein) stimulated by K+ ions was determined when sodium formate, ADP and inorganic phosphate were applied simultaneously. The results confirm the assumption about the dual role of hydrogenase 3, formate hydrogen lyase subunit, which is able to couple the reduction of protons to H2 and their translocation through a membrane with chemiosmotic synthesis of ATP.     

Accumulation of K+ in E. coli grown in aerobic conditions

The character of K+ accumulation in E. coli grown aerobilcally in the salt medium with succinate was studied. K+ uptake via the Trk system has Km 3.4 mM and Vmax 0.45 mM X g+1 X min-1. The initial rates of K+ uptake were not changes at different pH from 6.0 to 8.3 and temperature 17-37 degrees C. DCC did not block, protonophores and arsenate blocked the operation of Trk system. Valinomycin increased (or had no effect) K+ accumulation. K+ distribution is in good conformity with the measured membrane potential. The Trk system works at the utilization of lactic acid and glucose as well as of succinate. The Trk system is described. K+ ionophore by using the membrane potential and ATP regulates functioning of this system.     

pH-Responsive, posttranslational regulation of the Trk1 potassium transporter by the type 1-related Ppz1 phosphatase

Intracellular pH and K+ concentrations must be tightly controlled because they affect many cellular activities, including cell growth and death. The mechanisms of homeostasis of H+ and K+ are only partially understood. In the yeast Saccharomyces cerevisiae, proton efflux is mediated by the Pma1 H+-ATPase. As this pump is electrogenic, the activity of the Trk1 and -2 K+ uptake system is crucial for sustained Pma1p operation. The coordinated activities of these two systems determine cell volume, turgor, membrane potential, and pH. Genetic evidence indicates that Trk1p is activated by the Hal4 and -5 kinases and inhibited by the Ppz1 and -2 phosphatases, which, in turn, are inhibited by their regulatory subunit, Hal3p. We show that Trk1p, present in plasma membrane "rafts", physically interacts with Ppz1p, that Trk1p is phosphorylated in vivo, and that its level of phosphorylation increases in ppz1 and -2 mutants. Interestingly, both the interaction with and inhibition of Ppz1p by Hal3p are pH dependent. These results are consistent with a model in which the Ppz1-Hal3 interaction is a sensor of intracellular pH that modulates H+ and K+ homeostasis through the regulation of Trk1p activity.     

Relationship between formate hydrogen lyase and proton-potassium pump under heterolactic fermentation in Escherichia coli: functional multienzyme associations in the cell membrane

Anaerobically grown glucose-fermenting E. coli cells produce molecular hydrogen, acidify the medium and uptake potassium ions. It was shown that the H2 release and the proton-potassium exchange with the fixed (2H+/K+) stoichiometry of the initial DCC-sensitive fluxes were lost in mutants with the deleted fdhF gene or the hycA-H operon responsible for the biosynthesis of formate dehydrogenase H (FDH,H) or hydrogenase 3 (H3), respectively, which are the main components of the formate hydrogen lyase FHL(H). However, both processes occurred in mutants with the deleted hycE, hycF or hycG genes encoding the major and minor components of H3, respectively. The K+ uptake was sensitive to the osmotic shock resulting from glucose addition to the medium and decreased significantly in the presence of valinomycin. The H2 release and the 2H+/K+ exchange were absent in the mutant with the deleted hycB gene encoding the corresponding minor component of H3. This mutant acidified the medium and uptook K+ with Km typical for TrkA, but the stoichiometry of the DCC-inhibited fluxes was variable, and the K+ gradient between the cytoplasm and the medium in this mutant was lower than in the mutants lacking other minor components of H3. The results obtained suggest that the hycB gene product, FdhF and HycE, form probably the FHL(H) complex that directly interacts with the H+-ATPase complex F0F1 and the TrkA(H) system of K+ uptake. Such a multienzyme association is responsible for the H2 production and 2H+/K+ exchange. The major and other minor components of H3 have probably no direct role in the H2 production and 2H+/K+ exchange. H2 production by precursor's or hycE mutant's protoplasts treated with toluene was shown to occur upon addition of the thiol reagent dithiothreitol to the medium containing ATP, potassium ions, NAD+, and NADH. H2 production was inhibited by DCC. The quantity of available thiol groups in membrane vesicles of the precursor or the hycE, hycF or hycG mutants, in which the H2 production and 2H+/K+ exchange were observed, was larger than in other mutants. The number of SH groups decreased in the presence of DCC. These results indicate a significance of the thiol groups for the function of the proposed association.     

The action of sodium and potassium ions on the H+-ATPase activity of Escherichia coli K12 (lambda) membranes

The dependence of activity of H+-ATPase membranes of Escherichia coli K12 (lambda) grown anaerobically of potassium and sodium ions has been studied. The addition of K+ or Na+ to the reaction mixture causes an increase of H+-ATPase activity. The effect depended on conditions and keeping time of the preparation of membranes. The sensitivity of enzyme to potassium and sodium decreased with the rise of temperature from -20 degrees C to -4 degrees C and an increase of keeping time.     

The constitutive K+ pump in Serratia marcescens

Transport of K+ and H+ in the anaeronically and aerobically grown bacterium Serratia marcescens has been studied. The volumes of one cell of the anaerobically and aerobically grown bacterium were 3.7 X 10(-13) cm3 and 2.4 X 10(-13) cm3, respectively. Irrespective of the growth conditions the bacteria manifested the same respiration rate. However, the values of membrane potential for the anaerobically and aerobically grown bacterium were different and equal to -130 mV and -175 mV (interior negative), respectively, in the absence of an exogenic energy source. KCN + DCCD decreases delta psi down to almost zero in both species. DCCD alone decreases delta psi partially in anaerobes and increases delta psi in aerobes, whereas KCN alone reduces delta psi partially in both species. The introduction of glucose into the medium containing K+ reduces the absolute value of delta psi to [-160] mV in aerobes and to [-20] mV in anaerobes. The effect is not observed without external K+. In the presence of arsenate a delta psi is not reduced after the addition of glucose. At pH 7.5-7.8 the ATP level in aerobes grows notably faster than in anaerobes. The H+ extrusion becomes intensified when K+ uptake is activated by the increase in external osmotic pressure. Apparent Km and Vmax for K+ accumulation are 1.2 mM and 0.4 mM.min-1.g-1. The decrease of delta psi by glucose or KCN + DCCD have no effect on the K+ uptake whereas CCCP inhibits potassium accumulation. At the same time, arsenate stabilizes the delta psi value, but blocks K+ uptake. The accumulation of K+ correlates with the potassium equilibrium potential of -200 mV calculated according to the Nernst equation, whereas the delta psi measured was not more than [-25] mV. The calculated H+/ATP stoichiometry was 3.3 for aerobes. It was assumed that a constitutive K+ pump having a K+/ATP ratio equal to 2 or 3 operates in S. marcescens membranes.     

Effect of temperature on H+-K+ exchange in Escherichia coli bacteria during their anaerobic growth

The H(+)-K(+)-exchange in E.coli grown under anaerobic conditions at temperatures from 17 to 37 degrees C was studied. The Arrhenius plots for both the N,N'-dicyclohexylcarbodiimide-sensitive release of H+ and K+ uptake by cells transferred into a fresh medium containing a carbon source (glucose) are nonlinear. The activation energy values for the transport of these cations at different temperatures significantly differ. It is shown that as the temperature decreases, the accumulation of K+ by cells is reduced. In this process, the initial rate of K+ absorption through the TrkA system, the time of accumulation of these cations by cells and the osmosensitivity of K+ uptake substantially decrease. At temperatures below 20 degrees C, the absorption becomes insensitive to the secondary osmoshock. However, the stoichiometry of N,N'-dicyclohexylcar-bodiimide-sensitive cation fluxes remains unchanged and is equal to 2H+:K+. It is assumed that the H(+)-K(+)-exchange proceeds by the operation of an ensemble of oligomers, formed from the protomers of F0F1 and TrkA, which rearrange by the action of temperature, whereas F0F1 and TrkA in each protomer do not change.     

The presumed potassium carrier Trk2p in Saccharomyces cerevisiae determines an H+-dependent, K+-independent current

Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli.     

TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli.

The corrected sequence of the trkH gene of Escherichia coli predicts that the TrkH protein is a hydrophobic membrane protein of 483 amino acid residues, of which 41% are identical to those of the homologous and functionally analogous TrkG protein. These two proteins form the transmembrane component of the Trk system for the uptake of K+. Each protein alone is sufficient for high-level Trk activity. When Trk is assembled with the TrkG protein, Rb+ and K+ are transported with a Km near or below 1 mM; however, the Vmax for Rb+ is only about 7% of that for K+. When Trk is formed with TrkH, the affinities for both for K+ and Rb+ are somewhat lower, and the Vmax for Rb+ is only 1% of that for K+ transport. The kinetics of transport in strains with wild-type alleles at trkG and at trkH suggest that both products participate in transport.     

Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters

K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.     

The character of K+ uptake in anaerobically grown S. typhimurium

Tre character of K+ uptake in anaerobically grown S. typhimurium LT-2 is studied. In the alkaline media with glucose and moderate K+ activity these bacteria uptake K+ in two steps, the first of which has a high rate of K+ uptake, Km 2.1 mM and Vmax 0.44 mM/g. min and is sensitive to the medium osmolarity. Bacteria transfer from the media with high osmolarity to that with low one leads to a decrease of K+ uptake at the first step. The second increase of the medium osmolarity turns on the rapid K+ uptake only at alkaline pH. K+ uptake at the first step is inhibited by DCC and protonophores. In the absence of phosphate in the medium arsenate blocks K+ uptake at the first step, and when phosphate is available arsenate decreases K+ uptake. Valinomycin decreases the rate of K+ uptake. K+ uptake at the first step in S. typhimurium proceeds via Trk-like system which requires for K+ uptake both ATP and delta mu H+.     

Citrate transport in Klebsiella pneumoniae

Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.     

The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression

The yeast Ppz protein phosphatases and the Hal3p inhibitory subunit are important determinants of salt tolerance, cell wall integrity and cell cycle progression. We present several lines of evidence showing that these disparate phenotypes are connected by the fact that Ppz regulates K+ transport. First, salt tolerance, cell wall integrity and cell cycle phenotypes of Ppz mutants are dependent on the Trk K+ transporters. Secondly, Ppz mutants exhibit altered activity of the Trk system, as measured by rubidium uptake. Thirdly, Ppz mutants exhibit altered intracellular K+ and pH, as expected from H+ efflux providing electrical balance during K+ uptake. Our unifying picture of Ppz phenotypes contends that activation of Trk by decreased Ppz activity results in plasma membrane depolarization (reducing uptake of toxic cations), increased intracellular K+ and turgor (compromising cell integrity), and increased intracellular pH (augmenting the expression of pH-regulated genes and facilitating alpha-factor recovery). In addition to providing a coherent explanation for all Ppz-dependent phenotypes, our results provide evidence for a causal relationship between intracellular cation homeostasis and a potential cell cycle checkpoint.