Regulation of the plasma membrane potential in Pneumocystis carinii

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.     

Cell physiological aspects of the plasma membrane electrogenic H<Superscript>+</Superscript> pump

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H⁺) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H⁺ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H⁺ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H⁺ efflux supports the notion that the ion carried by the electrogenic ion pump is H⁺. The role of the H⁺ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H⁺ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the K_m value for the ATP of H⁺-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H⁺-ATPase in guard cells is presented. However, there are cases where H⁺-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H⁺-ATPase activity. Finally, modulation of H⁺-pumping or H⁺-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H⁺ pump.     

Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K(+) to this site can induce dephosphorylation of the phosphorylated E(1)P reaction cycle intermediate by a mechanism involving Glu(184) in the conserved TGES motif of the pump actuator domain. Our data identify K(+) as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H(+)/ATP coupling ratio. K(+)-induced dephosphorylation of E(1)P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E(1)P is accumulating.     

Plant proton pumps

Chemiosmotic circuits of plant cells are driven by proton (H(+)) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H(+) gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H(+)- pumps, namely the plasma membrane H(+)-ATPase, the vacuolar H(+)-ATPase and the vacuolar H(+)-PPase.     

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

Evidence for a plasma membrane proton pump in phloem cells of higher plants

Metabolic energy is required for the loading of sucrose into the phloem and translocation of sugars throughout the plant. The proton electrochemical gradient generated by a plasma membrane proton pump (H(+)-ATPase) is thought to provide energy for these processes. The plasma membrane H(+)-ATPase is encoded by a multigene family in Arabidopsis thaliana. Here we characterize the expression of isoform AHA3 (Arabidopsis H(+)-ATPase isoform 3). The AHA3 mRNA start site was mapped and 464 bp of the putative upstream regulatory region sequenced. A translational fusion of AHA3 to the beta-glucuronidase (GUS) reporter gene was constructed and used to generate transgenic Nicotiana and Arabidopsis plants. Using a histochemical stain, expression of the AHA3/GUS fusion was found predominantly in phloem cells of leaves, stems, roots, and flowers. Biochemical measurements of GUS activity in pith and vascular explants confirmed the histochemical localization. Our results support the hypothesis that a proton pump is present in phloem cells, possibly providing energy to drive plasma membrane cotransport systems required for phloem loading and translocation of photosynthates. In addition to AHA3/GUS expression in phloem, expression was observed in pollen and regions of the ovule, tissues whose physiological functions correlate with a requirement for high levels of solute transport.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

Significant differences between procyclic and bloodstream forms of Trypanosoma brucei in the maintenance of their plasma membrane potential

The plasma membrane potential (deltapsi) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of deltapsi in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H(+)-ATPase inhibitors (e.g. dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A1). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium-rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the deltapsi of the procyclic stages in a sodium-rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H(+)-ATPase inhibitor dicyclohexylcarbo-diimide, indicating the presence of an ouabain-sensitive sodium pump whose activity is masked by the H(+)-ATPase. Unlike procyclic forms, the deltapsi of bloodstream-stage trypomastigotes was markedly sensitive to extracellular Na+ and K+ concentrations. Thus, there are significant differences between procyclic and bloodstream forms in the maintenance of the deltapsi and in their permeability to cations.     

The plasma membrane proton pump ATPase: the significance of gene subfamilies

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a central role in transport across the plasma membrane. As a primary transporter, it mediates ATP-dependent H(+) extrusion to the extracellular space, thus creating pH and potential differences across the plasma membrane that activate a large set of secondary transporters. In several species, the H(+)-ATPase is encoded by a family of approximately 10 genes, classified into 5 gene subfamilies and we might ask what can this tell us about the concept, and the evolution, of gene families in plants. All the highly expressed H(+)-ATPase genes are classified into only two gene subfamilies, which diverged before the emergence of present plant species, raising the questions of the significance of the existence of these two well-conserved subfamilies and whether this is related to different kinetic or regulatory properties. Finally, what can we learn from experimental approaches that silence specific genes? In this review, we would like to discuss these questions in the light of recent data.     

Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. Its activity is regulated by a C-terminal autoinhibitory domain that can be displaced by phosphorylation and the binding of regulatory 14-3-3 proteins, resulting in an activated enzyme. To better understand the physiological consequence of this activation, we have analyzed transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H(+)-ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DeltaPMA4), generating a constitutively activated enzyme. Plants showing 4-fold higher expression of wtPMA4 than untransformed plants did not display any unusual phenotype and their leaf and root external acidification rates were not modified, while their in vitro H(+)-ATPase activity was markedly increased. This indicates that, in vivo, H(+)-ATPase overexpression is compensated by down-regulation of H(+)-ATPase activity. In contrast, plants that expressed DeltaPMA4 were characterized by a lower apoplastic and external root pH, abnormal leaf inclination, and twisted stems, suggesting alterations in cell expansion. This was confirmed by in vitro leaf extension and curling assays. These data therefore strongly support a direct role of H(+)-ATPase in plant development. The DeltaPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H(+)-ATPase is involved in salt tolerance.     

Blue light activates the plasma membrane H(+)-ATPase by phosphorylation of the C-terminus in stomatal guard cells.

The opening of stomata, which is driven by the accumulation of K(+) salt in guard cells, is induced by blue light (BL). The BL activates the H(+) pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H(+)-ATPase and that BL activates the H(+)-ATPase via phosphorylation. A pulse of BL (30 s, 100 micromol/m(2)/s) increased ATP hydrolysis by the plasma membrane H(+)-ATPase and H(+) pumping in Vicia guard cell protoplasts with a similar time course. The H(+)-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H(+)-ATPases on both serine and threonine residues in two isoproteins of H(+)-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H(+)-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H(+)-ATPases. These findings demonstrate that BL activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation.     

Lipid-dependent surface transport of the proton pumping ATPase: a model to study plasma membrane biogenesis in yeast

The proton pumping H+-ATPase, Pma1, is one of the most abundant integral membrane proteins of the yeast plasma membrane. Pma1 activity controls the intracellular pH and maintains the electrochemical gradient across the plasma membrane, two essential cellular functions. The maintenance of the proton gradient, on the other hand, also requires a specialized lipid composition of this membrane. The plasma membrane of eukaryotic cells is typically rich in sphingolipids and sterols. These two lipids condense to form less fluid membrane microdomains or lipid rafts. The yeast sphingolipid is peculiar in that it invariably contains a saturated very long-chain fatty acid with 26 carbon atoms. During cell growth and plasma membrane expansion, both C26-containing sphingolipids and Pma1 are first synthesized in the endoplasmatic reticulum from where they are transported by the secretory pathway to the cell surface. Remarkably, shortening the C26 fatty acid to a C22 fatty acid by mutations in the fatty acid elongation complex impairs raft association of newly synthesized Pma1 and induces rapid degradation of the ATPase by rerouting the enzyme from the plasma membrane to the vacuole, the fungal equivalent of the lysosome. Here, we review the role of lipids in mediating raft association and stable surface transport of the newly synthesized ATPase, and discuss a model, in which the newly synthesized ATPase assembles into a membrane environment that is enriched in C26-containing lipids already in the endoplasmatic reticulum. The resulting protein-lipid complex is then transported and sorted as an entity to the plasma membrane. Failure to successfully assemble this lipid-protein complex results in mistargeting of the protein to the vacuole.     

A cytosolic inhibitor of vacuolar H(+)-ATPases from mammalian kidney.

Regulation of the vacuolar H(+)-ATPase in organellar and transepithelial acidification has been attributed to the effects of the proton electrochemical gradient across the membrane or to changes in the number of proton pumps. We now report the identification and purification of a protein from bovine kidney cytosol that inhibits both ATPase activity and proton translocating activity of vacuolar H(+)-ATPases. Its relative molecular weight (M(r)) is 6300, similar to that for protein inhibitors of the mitochondrial F0F1-ATPase. The newly identified cytosolic inhibitor protein may participate in the physiologic regulation of the vacuolar H(+)-ATPase by suppressing activity directly.     

In vivo activation of plasma membrane H(+)-ATPase hydrolytic activity by complex lipid-bound unsaturated fatty acids in Ustilago maydis.

As an adaptation process to the growth retardation provoked by the presence of nonlethal concentrations of ergosterol biosynthesis inhibitors, Ustilago maydis alters the ratio of linoleic to oleic acid bound to plasma membrane complex lipids [Hernández, A., Cooke, D.T., Lewis, M. & Clarkson, D.T. (1997) Microbiology 143, 3165-3174]. This alteration increases plasma membrane H(+)-ATPase hydrolytic activity. Activation of H(+)-ATPase by the linoleic/oleic acid proportion is noncompetitive, nonessential and only involves changes in the maximum velocity of the pump. Optimum pH, affinity to MgATP and constants for the inhibition by vanadate and erythrosin B remain unchanged. This all indicates that activation of plasma membrane H(+)-ATPase by unsaturated fatty acids differs clearly from glucose-induced activation observed in yeast. Also, it is a physiologically relevant event similar to other, as yet uncharacterized, changes in plasma membrane H(+)-ATPase hydrolytic activity observed in plants and fungi, as part of an adaptation process to different stress conditions.     

Cyclic stretch translocates the alpha2-subunit of the Na pump to plasma membrane in skeletal muscle cells in vitro

The Na+-K+-ATPase and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all skeletal muscle cells and thus is essential for cell survival and function. In our previous study, cyclic stretch activated the Na pump in cultured skeletal muscle cells. Presently, we investigated whether this stimulation was the result of translocation of Na+-K+-ATPase from endosomes to the plasma membrane, and also evaluated the role of phosphatidylinositol 3-kinase (PI 3-kinase), the activation of which initiated vesicular trafficking and targeting of proteins to specific cell compartments. Skeletal muscle cells were stretched at 25% elongation continuous for 24h using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+-K+-ATPase alpha1- and alpha2-subunit protein. The results showed stretch increased Na+-K+-ATPase alpha1- and alpha2-subunit protein expression in plasma membrane fractions and decreased it in endosomes. The alpha2-subunit had a more dynamic response to mechanical stretch. PI 3-kinase inhibitors (LY294002) blocked the stretch-induced translocation of the Na+-K+-ATPase alpha2-subunit, while LY294002 had no effect on the transfer of alpha1-subunit. We concluded that cyclic stretch mainly stimulated the translocation of the alpha2-subunit of Na+-K+-ATPase from endosomes to the plasma membrane via a PI 3-kinase-dependent mechanism in cultured skeletal muscle cells in vitro, which in turn increased the activity of the Na pump.     

Electric field strength of membrane lipids from vertebrate species: membrane lipid composition and Na+-K+-ATPase molecular activity

Intramembrane electric field strength is a very likely determinant of the activity of ion-transporting membrane proteins in living cells. In the absence of any transmembrane electrical potential or surface potential, its magnitude is determined by the dipole potential of the membrane's lipid components and their associated water of hydration. Here we have used a fluorometric method to quantify the dipole potential of vesicles formed from lipids extracted from kidney and brain of 11 different animal species from four different vertebrate classes. The dipole potential was compared with the fatty acid composition and with the Na(+)-K(+)-ATPase molecular activity of each preparation. The magnitude of the dipole potential was found to be relatively constant across all animal species, i.e., 236-334 mV for vesicles prepared from the total membrane lipids and 223-256 mV for phospholipids alone. The significantly lower value for phospholipids alone is potentially related to the removal of cholesterol and/or other common soluble lipid molecules from the membrane. Surprisingly, no significant dependence of the dipole potential on fatty acid composition was found. This may, however, be due to concomitant compensatory variations in lipid head group composition. The molecular activity of the Na(+)-K(+)-ATPase was found to increase with increasing dipole potential. The fact that the dipole potential is maintained at a relatively constant value over a wide range of animal species suggests that it may play a fundamental role in ensuring correct ion pump conformation and function within the membrane.     

Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.