Acidification of endocytic vesicles by an ATP-dependent proton pump

One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled alpha 2- macroglobulin (F-alpha 2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-alpha 2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(beta, gamma- imido)triphosphate (AMP-PNP) could support acidification. The ATP- dependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn2+ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS). We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP- dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.     

Calcium activates an electrogenic proton pump in neurospora plasma membrane

Calcium ionophoresis into coenocytic cells of Neurospora crassa activates the plasma membrane proton pump as measured by current-voltage analysis. This is direct evidence that intracellular calcium regulates the activity of a key transport enzyme found in higher plants and fungi.     

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80 mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H⁺-ATPase. However, there was no effect of NaCl on water permeability (P_f) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H⁺-ATPase activity, thereby helping to reduce partially the Na⁺ concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H⁺-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.     

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.     

The role of proton pump and potassium channels of plasma membrane in regulation of cellulose synthesis in plants

A study was made of the influence of two activators of plasma membrane proton pump [indole-3-acetic acid (IAA) and fusicoccin (EC)] and of the blocker of potassium channels of outward direction [tetraethylammonium chloride (TEA)] on exogenous [U-14C]glucose incorporation into cellulose fraction of cell wall, and on the value of plasmalemma membrane potential. It has been shown that IAA and FC exert different influences on the intensity of [U-14C]glucose incorporation into cellulose fraction: IAA activates, while FC inhibits incorporation intensity. A conclusion is made that differences in affects of IAA and FC on the intensity of cellulose synthesis at the plasma membrane level may be due to the fact that the activating effect of IAA on plasma membrane proton pump involves activation of the inward direction potassium channels, whereas that of FC, on the contrary, is associated with their blocking. Under the action of TEA, the intensity of incorporation of radioactively labeled glucose was increased. Apparently, the role of plasma membrane in regulation of the intensity of cellulose synthesis may be associated with not only the activity of proton pump on plasma membrane, but also the functional condition of potassium channels of this membrane.     

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).     

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.     

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.     

A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages

Phagocytes generate large amounts of metabolic acid during activation. Therefore, the presence of a conductive pathway capable of H+ extrusion has been suggested (Henderson, L. M., J. B. Chappell, and O. T. G. Jones. 1987. Biochemical Journal. 246:325-329). In this report, electrophysiological and fluorimetric methods were used to probe the existence of a H+ conductance in murine peritoneal macrophages. In suspended cells, recovery of the cytosolic pH (pHi) from an acid-load in Na+ and HCO3(-)-free medium was detectable in depolarizing but not in hyperpolarizing media. The rate of alkalinization was potentiated by the rheogenic ionophore valinomycin. These findings are consistent with the existence of a conductive H+ (equivalent) pathway. This notion was confirmed by patch-clamping and fluorescence ratio measurements of single adherent cells. When voltage was clamped in the whole-cell configuration, depolarizing pulses induced a sizable outward current which was accompanied by cytosolic alkalinization. Several lines of evidence indicate that H+ (equivalents) carry this current: (a) the conductance was unaffected by substitution of the major ionic constituents of the intra-and/or extracellular media, (b) the reversal potential of the tail currents approached the H+ equilibrium potential; and (c) the voltage-induced currents and pHi changes were both Zn2+ sensitive and had similar time course and potential dependence. The peak whole-cell current displayed marked outward rectification and was exquisitely H+ selective. At constant voltage, the H+ permeability was increased by lowering pHi but was inhibited by extracellular acidification. Together with the voltage dependence of the conductance, these features ensure that H+ extrusion can occur during activation, while potentially deleterious acid uptake is precluded. The properties of the conductance appear ideally suited for pHi regulation during phagocyte activation, because these cells undergo a sustained depolarization and an incipient acidification when stimulated. Comparison of the magnitude of the current with the amount of metabolic acid generated during macrophage activation indicates that the conductance is sufficiently large to contribute to the H+ extrusion required for maintenance of pHi.     

Modeling a conformationally sensitive region of the membrane sector of the fungal plasma membrane proton pump

A molecular model for transmembrane segments 1 and 2 from the fungal proton pumping ATPase has been developed, and this structure is predicted to form a helical hairpin loop structure in the membrane. This region was selected because it is highly conformationally active and is believed to be an important site of action for clinically important therapeutics in related animal cell enzymes. The hairpin loop is predicted to form an asymmetric tightly packed structure that is stabilized by an N-cap between D140 and V142, by hydrogen bonding between residues in the turn region and the helices, and by - interactions between closely apposed aromatic residues. A short four-residue S-shaped turn is stabilized by hydrogen bonding but is predicted to be conformationally heterogeneous. The principal effect of mutations within the hairpin head region is to destabilize the local close packing of side groups which disrupts the pattern of hydrogen bonding in and around the turn region. Depending on the mutation, this causes either a localized or a more global distortion of the primary structure in the hairpin region. These altered structures may explain the effects of mutations in transmembrane segments 1 and 2 on ATP hydrolysis, sensitivity to vanadate, and electrogenic proton transport. The conformational sensitivity of the hairpin structure around the S-turn may also account for the effects of SCH28080 and possibly ouabain in blocking ATPase function in related animal cell enzymes. Finally, the model of transmembrane segments 1 and 2 serves as a template to position transmembrane segments 3 and 8. This model provides a new view of the H⁺-ATPase that promotes novel structure/function experimentation and could serve as the basis for a more detailed model of the membrane sector of this enzyme.     

Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles

Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K_m of about 0.23 millimolar for MgATP, is only slightly affected by K⁺ or Cl⁻ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO₃⁻ > Cl⁻) and has a K_m of about 0.7 millimolar. Auxins decrease the K_m to about 0.35 millimolar, with no significant effect on the V_max, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.     

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Synthesis of phosphatidylcholines in rat hepatocytes. Possible regulation by norepinephrine via an alpha-adrenergic mechanism

The effect of norepinephrine on phosphatidylcholine and phosphatidylethanolamine formation was investigated in short-term incubations with freshly isolated rat hepatocytes. In the presence of dl-propranolol, norepinephrine decreases the incorporation of [methyl-14C]choline into phosphatidylcholines in a dose-dependent manner. At a concentration of 50 microM, norepinephrine (plus 20 microM propranolol) inhibits the incorporation of [methyl-14C]choline over a wide range of choline concentrations (59% inhibition at 5 microM choline; 34% inhibition at 1 mM choline). Norepinephrine also decreases the incorporation rates of [1-14C]palmitic acid and [1-14C]oleic acid into phosphatidylcholines. The effect of norepinephrine is mediated through an alpha-adrenergic receptor. Norepinephrine (plus propranolol) does not decrease the uptake or phosphorylation rate of [methyl-14C]choline. Pulse-label and pulse-chase studies indicate that the conversion rate of phosphocholine to CDP-choline, catalyzed by CTP:phosphocholine cytidylyltransferase, is diminished by norepinephrine. In contrast with the inhibitory effect of norepinephrine on phosphatidylcholine synthesis, this hormone stimulates the formation of phosphatidylethanolamines from [1,2-14C]ethanolamine. This increased incorporation rate is apparent at ethanolamine concentrations above 25 microM. A combination of norepinephrine and propranolol decreases, however, the synthesis of phosphatidylcholines from [1,2-14C]ethanolamine. The results indicate that alpha-adrenergic regulation dissociates the synthesis of phosphatidylcholines from that of phosphatidylethanolamines.