The yeast mutant vps5Delta affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg2+/H+ exchange activity.

Growth of the yeast vacuolar protein-sorting mutant vps5Delta affected in the endosome-to-Golgi retromer complex was more sensitive to Mg2+-limiting conditions than was the growth of the wild-type (WT) strain. This sensitivity was enhanced at acidic pH. The vps5Delta strain was also sensitive to Al3+, known to inhibit Mg2+ uptake in yeast cells. In contrast, it was found to be resistant to Ni2+ and Co2+, two cytotoxic analogs of Mg2+. Resistance to Ni2+ did not seem to result from the alteration of plasma-membrane transport properties because mutant and WT cells displayed similar Ni2+ uptake. After plasma-membrane permeabilization, intracellular Ni2+ uptake in vps5Delta cells was 3-fold higher than in WT cells, which is consistent with the implication of the vacuole in the observed phenotypes. In reconstituted vacuolar vesicles prepared from vps5Delta, the rates of H+ exchange with Ni2+, Co2+, and Mg2+ were increased (relative to WT) by 170%, 130%, and 50%, respectively. The rates of H+ exchange with Ca2+, Cd2+, and K+ were similar in both strains, as were alpha-mannosidase and H+-ATPase activities, and SDS/PAGE patterns of vacuolar proteins. Among 14 other vacuolar protein-sorting mutants tested, only the 8 mutants affected in the recycling of trans-Golgi network membrane proteins shared the same Ni2+ resistance phenotype as vps5Delta. It is proposed that a trans-Golgi network Mg2+/H+ exchanger, mislocalized to vps5Delta vacuole, could be responsible for the phenotypes observed in vivo and in vitro.

Passive nitrate transport by root plasma membrane vesicles exhibits an acidic optimal pH like the H(+)-ATPase.

The net initial passive flux (J(Ni)) in reconstituted plasma membrane (PM) vesicles from maize (Zea mays) root cells was measured as recently described (P. Pouliquin, J.-P. Grouzis, R. Gibrat ¿1999 Biophys J 76: 360-373). J(Ni) in control liposomes responded to membrane potential or to NO(3)(-) as expected from the Goldman-Hodgkin-Katz diffusion theory. J(Ni) in reconstituted PM vesicles exhibited an additional component (J(Nif)), which was saturable (K(m) for NO(3)(-) approximately 3 mM, with J(Nifmax) corresponding to 60 x 10(-9) mol m(-2) s(-1) at the native PM level) and selective (NO(3)(-) = ClO(3)(-) > Br(-) > Cl(-) = NO(2)(-); relative fluxes at 5 mM: 1:0.34:0.19). J(Nif) was totally inhibited by La(3+) and the arginine reagent phenylglyoxal. J(Nif) was voltage dependent, with an optimum voltage at 105 mV at pH 6.5. The activation energy of J(Nif) was high (129 kJ mol(-1)), close to that of the H(+)-ATPase (155 kJ mol(-1)), and J(Nif) displayed the same acidic optimal pH (pH 6.5) as that of the H(+) pump. This is the first example, to our knowledge, of a secondary transport at the plant PM with such a feature. Several properties of the NO(3)(-) uniport seem poorly compatible with that reported for plant anion channels and to be attributable instead to a classical carrier. The physiological relevance of these findings is suggested.

Reconstitution of an electrogenic auxin transport activity mediated by Arabidopsis thaliana plasma membrane proteins.

Plasma membrane proteins from Arabidopsis thaliana leaves were reconstituted into proteoliposomes and a K+ diffusion potential was generated. The resulting ionic fluxes, determined in the presence of the plant hormone auxin (indole-3 acetic acid), showed an additional electrogenic and saturable component, with a K(M) of 6 microM. This flux was neither detected in liposomes in the presence of indole-3 acetic acid, nor in proteoliposomes in the presence of an inactive auxin analog and was completely inhibited by 3 microM naphtylphthalamic acid, a specific inhibitor of the auxin efflux carrier. The efficiency of the reconstituted carrier and the mechanism of its regulation by naphtylphthalamic acid are discussed.

Electrophysiological study with oxonol VI of passive NO3- transport by isolated plant root plasma membrane.

In contrast to animal cells, plant cells contain approximately 5-50 mM nitrate in cytosol and vacuole. The lack of specific spectroscopic probes, or suitable isotopes, impedes in vitro studies of NO3- transport. Reconstitution of root cell plasma membrane (PM) proteins in mixed soybean lipid:egg phosphatidylcholine allowed for the generation of large K+-valinomycin diffusion potentials (Em), monitored with the oxonol VI dye. Nevertheless, Em was restricted to approximately 130 mV by capacitor properties of biological membranes. This caused an increasing discrepancy at higher K+-Nernst potentials used for calibration. Therefore, Em was determined directly from the fluorescence of the dye free in buffer, bound at zero Em, and bound upon Em generation. Then, an electrophysiological analysis of the NO3--dependent dissipation rate of Em gave the net passive flux (JN) and the permeability coefficient to NO3- (PN). The plant root cell PM exhibited a strikingly large PN (higher than 10(-9) m s-1) at high Em (90-100 mV) and pH 6.5. At low Em (50-60 mV) and pH 7.4, PN decreased by 70-fold and became similar to that of the lipid bilayer. This agreed with the previous observation that 15 mM NO3- short-circuits the plant root PM H+-ATPase at its optimal pH of 6.5.

In vitro characterization of iron-phytosiderophore interaction with maize root plasma membranes: evidences for slow association kinetics.

As an attempt to characterize iron(III)-phytosiderophore transport across plant membranes in vitro, a rapid filtration approach was set up in which plasma membrane vesicles from maize roots were incubated with 55Fe-labelled deoxymugineic acid (DMA). Fe-DMA, and not Fe-EDTA, could associate with plasma membrane vesicles. The rate of Fe-DMA association decreased with a half time of 15 min. The initial Fe-DMA association rate, estimated from the amount of Fe-DMA associated after 10 min incubation, exhibited a saturation curve as a function of Fe-DMA concentration. This curve could be satisfactorily fitted to the Michaelis-Menten model (KM=600 nM, Vmax=2 nmol min-1 mg-1 protein). The association rate of Fe-DMA with control liposomes remained negligible and constant in a pH range from 4 to 8, whereas it strongly increased at acidic pH with plasma membrane vesicles. However, the specific association of Fe-DMA to root plasma membrane could not be explained by a vesicle-filling process because: (i) lowering the vesicle volume by decreasing the osmotic potential of the assay medium with sorbitol did not decrease 55(Fe) labelling of the vesicles, (ii) creating inside-out vesicles by a Brij-58 treatment had almost no effect on Fe-DMA association to vesicles, (iii) 55(Fe) labelling is reversible by EDTA and excess free DMA, and (iv) 55(Fe) labelling was the same using plasmalemma vesicles prepared either from wild type maize or from the ys1 maize mutant deficient in iron-phytosiderophore transport. A model is proposed to account for the observed Fe-DMA association as the result of very slow binding kinetics onto membrane proteins. This model was validated by its ability to describe quantitatively both Fe-DMA association as a function of time and of substrate concentration. A prediction of the model was that association of Fe-DMA to plasma membranes might overcome a high activation energy barrier. Indeed, the Arrhenius plot for the association rate constant was linear with an activation energy of 64 kJ mol-1.

In vitro study of passive nitrate transport by native and reconstituted plasma membrane vesicles from corn root cells.

Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H+)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K+ diffusion gradient. Consequently, the H(+)-pumping activity of the (H+)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO3- to electrically short-circuit the (H+)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K(m) approximately 5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO3- > I- approximately ClO3- approximately Br- > Cl- approximately NO2- > Iminodiacetate approximately SO4(2-)). Passive NO3- transport was also determined, independently of the (H+)ATPase, from the NO3(-)-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K(m) for NO3- and should be suitable to continue the functional and biochemical characterization of the NO3- transport system.

The plant inorganic pyrophosphatase does not transport K+ in vacuole membrane vesicles multilabeled with fluorescent probes for H+, K+, and membrane potential.

It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J. M., Poole, R. J., Rea, P. A., and Sanders, D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11701-11705). Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification. We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential. This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+. Proton pumping occurred in response to pyrophosphate addition in the absence of K+. No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated. The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin. No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase. We conclude that PPase in native vacuolar membrane vesicles does not transport K+.

Solubilization and Reconstitution of the Mg2+/2H+ Antiporter of the Lutoid Tonoplast from Hevea brasiliensis Latex.

The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou, R. Gibrat, C. Brugidou, P. Trouslot, J.d'Auzac [1992] Plant Physiol 100: 255-260) was solubilized by octylglucoside and reconstituted into soybean liposomes using the detergent dilution method. Magnesium efflux or influx experiments were used to generate a H+ influx or efflux, respectively, monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine. Both experiments gave saturable H+ fluxes as a function of internal or external Mg2+ concentrations with similar kinetic parameters Km and Vmax. The Km value for Mg2+ (about 2 mM) was identical to that previously found in lyophilized-resuspended lutoid (reference therein), whereas the Vmax value was 14-fold higher. Since only 10% of the initial proteins were recovered in proteoliposomes, and electrophoretic patterns of the two kinds of vesicles differed significantly, it was inferred that the increase in Vmax was due essentially to an enrichment of the protein antiporter in the reconstituted fraction, owing to a selective effect of octylglucoside at both solubilization and reconstitution steps. None of the various divalent cations used could dissipate the pH gradient of control liposomes of soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas a rapid dissipation of the pH gradient was observed with reconstituted proteoliposomes from lutoid proteins, with the cation selectivity sequence Zn2+ > Cd2+ > Mg2+ in the millimolar concentration range. The divalent ions Ca2+, Ba2+, and Mn2+ were incapable of generating a H+ efflux in reconstituted proteoliposomes, whereas both Mg2+/H+ and Ca2+/H+ exchanges were observed in lyophilized-resuspended lutoids. Therefore, the lutoid membrane seems to contain separate Mg2+/H+ and Ca2+/H transport systems, the latter being eliminated during the solubilization/reconstitution of lutoid membrane proteins.

A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles.

The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.

Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes.

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis.

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg(2+) diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg(2+). These results suggest the presence of a Mg(2+)/H(+) antiporter. The maximum Mg(2+)/H(+) exchange rate was observed at pH 8.5. The K(m) values for Mg(2+) (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K(+) and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg(2+)/H(+) exchange was electroneutral. Mg(2+)/H(+) exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K(m) for Mg(2+) are similar to those reported for the Mg(2+)/2Na(+) antiporter in animals cell. These data are consistent with the existence of a Mg(2+)/2H(+) antiporter in a plant tonoplast.

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer.

The purified (H+)ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H(+)-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H+)ATPase molecules were closely associated with liposomes, exhibiting a high H(+)-pumping activity (150,000% quenching min-1.mg-1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H+)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H+)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles ("inside-out" orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H+)ATPase could also functionally insert into bilayer from PC:PE:PG or PC:PE:PI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H+)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H+)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation.

Potassium Stimulation of Corn Root Plasmalemma ATPase : II. H-Pumping in Native and Reconstituted Vesicles with Purified ATPase.

The stimulation by K(+) of the initial rate of H(+)-pumping by ATPase was studied in native plasmalemma (Zea mays L. var Mona) vesicles and in reconstituted vesicles with enzyme purified on a glycerol gradient. In reconstituted vesicles, a very high H(+)-pumping rate (200,000% quenching per minute per milligram protein) was obtained with 9-amino-6-chloro-2-methoxyacridine provided that the pump was short-circuited by K(+)-valinomycin. A constant ionic strength was used to prevent indirect stimulation by the electrostatic effects of K(+) salts. Indirect stimulation of H(+)-pumping by the short-circuiting effect of internal K(+), could be abolished by using the permeant anions NO(3) (-) and Br(-) in native, but not in reconstituted vesicles. In both materials, half-stimulation of the H(+)-pumping by K(+) was observed at about 5 millimolar. The same stimulation was obtained when K(+) was present only in the external solution or when it was present both outside and inside the vesicles. It was concluded that the stimulating effect of K(+) on the H(+)-pumping evidenced in these experiments on both native and reconstituted vesicles was due to a direct effect of the cation on the cytoplasmic face of the ATPase. These results are discussed within the context of the hypothesis of an active K(+) transport driven by the ATPase through a direct H(+)/K(+) exchange mechanism.

Potassium stimulation of corn root plasmalemma ATPase : I. Hydrolytic activity of native vesicles and purified enzyme.

Potassium stimulation of the plasmalemma (Zea mays L. var Mona) was studied by using a constant ionic strength to prevent indirect stimulation by the electrostatic effect of K(+) salts. The transmembrane electrochemical H(+) gradient was eliminated by using gramicidin. In these conditions, K(+) stimulation was attributable to a direct effect of the cation on plasmalemma proteins. We used both native vesicles isolated on a sucrose cushion, and solubilized and purified ATPase from phase-partitioned plasmalemma, according to the method of T. Nagao, W. Sasakawa, and T. Sugiyama ([1987] Plant Cell Physiol 28: 1181-1186). The purified enzyme had a high specific activity (15 micromoles per minute per milligram protein), but was only about 20% stimulated by K(+). In both preparations, potassium (in the range around 1 millimolar) specifically decreased two-fold the vanadate inhibition constant, and increased the maximum rate of ATP hydrolysis. In plasmalemma vesicles, the Eadie-Scatchard graph of the K(+)-dependent ATPase activity as a function of K(+) concentration was linear only at constant ionic strength. The purified ATPase preparation appeared as two closely spaced bands in the 100 kilodalton region with isoelectric point about 6.5. Nevertheless, this biochemical heterogeneity seems unlikely to be related to K(+) stimulation, since K(+) modified neither the pH optimum of the activity (pH 6.5) nor the monophasic kinetics of the vanadate inhibition, in both native plasmalemma and purified enzyme preparation.

Electrostatic analysis of effects of ions on the inhibition of corn root plasma membrane Mg2+-ATPase by the bivalent orthovanadate.

The microelectrophoretic mobility of corn root plasma membranes and the inhibition of the Mg+2-ATPase by vanadate were investigated under different ionic conditions. The Mg2+-ATPase was uncompetitively inhibited and a 10-fold variation of the apparent inhibition constant was observed, depending on the addition of K+ and Mg2+. The determination of the zeta potential indicated that a 5-fold decrease of the apparent inhibition constant was due to aspecific electrostatic interactions of the vanadate anion and the negative charge of the membrane. The screening and masking effects of 6 mM free Mg2+ totally abolished electrostatic interactions and allowed the direct determination of the intrinsic vanadate inhibition constant (KIi). On the other hand, a specific, non-electrostatic, effect of K+ caused a 2-fold decrease of the inhibition constant in addition to the electrostatic effect. Finally, the electrostatic analysis indicates that the Mg2+-ATPase is inhibited by the monomeric bivalent anion HVO4(2-).

Preparation of Corn Root Plasmalemma with Low Mg-ATPase Latency and High Electrogenic H Pumping Activity after Phase Partitioning.

Crude plasma membranes of corn (Zea mays L.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H(+)-transport, was purified by phase partitioning. The percentage of inside-out vesicles (10%) was determined from the Mg-ATPase latency, revealed with lysophosphatidylcholine. A Triton X-100 treatment described previously (JP Grouzis, R Gibrat, J Rigaud, C Grignon 1987 Biochim Biophys Acta 903: 449-464) was applied to phase-partitioned plasma membranes. The percentage of catalytic sites freely accessible to Mg-ATP increased to 50% after Triton X-100 treatment. Treated vesicles remained capable of electrogenic H(+)-pumping, as demonstrated by Mg:ATP-dependent quinacrine fluorescence quenching and oxonol absorbance shift. As expected from the large increase of the catalytic sites accessibility, increases of the dye responses were observed. Concanavalin A binding was estimated from microelectrophoretic measurements of individual vesicles. Statistical analysis of concanavalin A binding and Mg-ATPase latency suggest that treated membranes have lost their asymmetric structure.

A procedure for estimating the surface potential of charged or neutral membranes with 8-anilino-1-naphthalenesulphonate probe. Adequacy of the Gouy-Chapman model.

Using the fluorescent anion 8-anilino-1-naphthalenesulphonate (ANS) for determining the membrane surface potential necessitates that the intrinsic affinity constant Ki for the ANS sites be known. Two methods are presented which do not rely on a determination of Ki at high ionic strength. They are respectively applied to neutral membranes (egg phosphatidylcholine liposomes) and highly charged natural ones (horse bean microsomes and liposomes from their phospholipids). The value of Ki appears to be insensitive to the level of occupancy of the sites, the KCl concentration and the pH in large ranges. Furthermore, the classical Gouy-Chapman model seems to describe correctly the whole set of data, provided apparent mean molecular areas larger than the published crystallographic ones are admitted.