Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots: II. Sensitivity of Development of the Augmented Uptake Potential to Toxin and Inhibitors of Protein Synthesis

Basal K⁺ uptake in the root midzone region (cm 2 + 3 + 4) of N and T cytoplasmic versions of each of four maize inbreds was equally sensitive to the toxin(s) of Helminthosporium maydis, race T. Basal K⁺ uptake in the root apex (0-1 cm) and augmented K⁺ uptake in the root midzone were more toxin-sensitive in inbreds W64A(T) and Mo17(T) than in inbreds W64A(N) and Mo17(N). This differential response of N and T cytoplasms to toxins was not found for corresponding cytoplasms of inbreds WF9 and B37.     

Potassium and sodium absorption kinetics in roots of two tomato species : lycopersicon esculentum and lycopersicon cheesmanii

Excised roots of the tomato species, Lycopersicon esculentum Mill. cv Walter (the commercial species) and of Lycopersicon cheesmanii ssp. minor (Hook.) C.H. Mull. (a wild species from the Galapagos Islands), were used in comparative studies of their absorption of K⁺ and Na⁺. Uptake of ⁸⁶Rb-labeled K⁺ and ²²Na-labeled Na⁺ by excised roots of `Walter' and L. cheesmanii varied as a function of genotype and tissue pretreatment with or without K<sup>+</sup>. Excised roots of `Walter' consistently absorbed more ⁸⁶Rb-labeled K⁺ than those of L. cheesmanii. Absorption of K⁺ from solutions ranging from 0.01 to 0.2 millimolar KCl showed saturation kinetics in both K⁺-pretreated and K⁺-depleted roots of `Walter,' and for K<sup>+</sup>-depleted roots of L. cheesmanii. K<sup>+</sup>-pretreated roots of L. cheesmanii had exceedingly low rates of K<sup>+</sup> uptake with strikingly different, linear kinetics. Pretreatment with K<sup>+</sup> caused a decrease in rates of K<sup>+</sup> uptake in both genotypes. Potassium depleted roots of L. cheesmanii absorbed Na<sup>+</sup> at a greater rate than those of `Walter,' whereas K⁺-pretreated roots of `Walter' absorbed Na⁺ at a greater rate than those of L. cheesmanii. The results confirm and extend previous conclusions to the effect that closely related genotypes may exhibit widely different responses to the two alkali cations, K⁺ and Na⁺.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Bicarbonate Fixation and Malate Compartmentation in Relation to Salt-induced Stoichiometric Synthesis of Organic Acid

The relationship of malate synthesis to K⁺ absorption from solutions of K₂SO₄ and KHCO₃ was compared in nonvacuolate barley (Hordeum vulgare) root tips and whole excised roots. The comparison has permitted separation of the process which evokes organic acid synthesis from that which leads to stoichiometry between net acid equivalents formed and excess K⁺ absorbed from K₂SO₄, on the one hand, and total K⁺ absorbed from KHCO₃, on the other. Both in tips and in roots K⁺ uptake from 20 mN salt solution exceeds malate synthesis in the first hour. In vacuolate roots the expected stoichiometry is achieved with time. When root tips are transferred to dilute CaSO₄, malate is rapidly metabolized, and K⁺ is lost to the solution. By contrast, in excised whole roots the malate level remains unchanged, the salt-induced organic acid presumably being retained in the vacuole. In excised roots malonate leads to a marked drop in malate levels in untreated roots as well as in roots which have experienced salt-induced net malate synthesis. In consequence, it is contended that malonate makes available normally sequestered vacuolar malate.     

Potassium transport in salt-stressed barley roots

Salinization of the medium inhibits both K⁺ uptake by excised barley (Hordeum vulgare L.) roots and K⁺ release from their stele, as measured by short-term ⁸⁶Rb uptake and xylem exudation, respectively. Although inhibition was not specific to chloride, mannitol caused a different response from that of inorganic sodium salts, indicating that inhibition was at least partly the result of an ion effect. In roots previously exposed to low levels of NaCl, NaCl stress directly affected stelar K⁺ release, whereas in low-sodium roots stelar K⁺ release was much less salt-sensitive than K⁺ uptake.Abbreviation chCl choline chloride     

Potassium Transport in Corn Roots : III. Perturbation by Exogenous NADH and Ferricyanide

It has recently been reported that plasmalemma electron transport may be involved in the generation of H⁺ gradients and the uptake of ions into root tissue. We report here on the influence of extracellular NADH and ferricyanide on K⁺ (⁸⁶Rb⁺) influx, K⁺ (⁸⁶Rb⁺) efflux, net apparent H⁺ efflux, and O₂ consumption in 2-centimeter corn (Zea mays [A632 × Oh43]) root segments and intact corn roots. In freshly excised root segments, NADH had no effect on O₂ consumption and K⁺ uptake. However, after the root segments were given a 4-hour wash in aerated salt solution, NADH elicited a moderate stimulation in O₂ consumption but caused a dramatic inhibition of K⁺ influx. Moreover, net apparent H⁺ efflux was significantly inhibited following NADH exposure in 4-hour washed root segments.

Exogenous ferricyanide inhibited K⁺ influx in a similar fashion to that caused by NADH, but caused a moderate stimulation of net H⁺ efflux. Additionally, both reagents substantially altered K⁺ efflux at both the plasmalemma and tonoplast.

These complex results do not lend themselves to straightforward interpretation and are in contradiction with previously published results. They suggest that the interaction between cell surface redox reactions and membrane transport are more complex than previously considered. Indeed, more than one electron transport system may operate in the plasmalemma to influence, or regulate, a number of transport functions and other cellular processes. The results presented here suggest that plasmalemma redox reactions may be involved in the regulation of ion uptake and the `wound response' exhibited by corn roots.

     

Loss of recovery capacity of plasmalemma k influx after cutting in chlorsulfuron pretreated maize roots

Active K⁺ influx was studied in apical segments from maize (Zea mays L., hybrid lines XL 342) and pea (Pisum sativum L. var Laxton superbo) seedlings pretreated with the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) aminocarbonyl]benzenesulfonamide).

Even though both plants were sensitive to chlorsulfuron, a strong inhibition of K⁺ uptake only was evident in maize root segments after 12 hours pretreatment with 10 micromolar chlorsulfuron. The inhibition was revealed only when maize root segments were washed for 2 hours before uptake measurements. This was done in order to recover K⁺ influx inhibited by cutting injury. Consequently, we demonstrated that roots from chlorsulfuron pretreated maize seedlings lost the capacity to recover from cutting injury by washing. By contrast, K⁺ influx in pea roots was not inhibited by chlorsulfuron because pea roots notoriously do not exhibit the `washing' effect.

     

Influence of Excision and Aging upon K+ Influx into Barley Roots: Recovery or Enhancement? 1

The influx of K⁺ from ⁸⁶Rb-labeled solutions in the concentration range 0.008 to 0.2 mm into roots of intact plants and excised roots of barley plants (Hordeum vulgare [L.]) previously grown in 5 mm CaSO₄ (low K⁺ roots) or 0.5 mm CaSO₄ plus 5 mm KCl (high K⁺ roots) was measured. A consistent observation of these experiments was a substantial reduction of influx (usually by about 50%) following excision. The possible leakage of K⁺ into the medium and subsequent dilution of specific activity of labeled solutions was eliminated as an explanation for influx reduction in excised low K⁺ roots. Reduction of transpirational rates was also without effect upon influx into low K⁺ roots. Excision followed by 2 hours aging in 0.5 mm CaSO₄ solution revealed that influx values recovered within the 2 hours to the values obtained in intact roots. It is concluded that much of the literature which describes the enhancement of ion uptake following excision actually describes excision damage followed by recovery.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.     

Complexity of potassium acquisition: How much flows through channels?

The involvement of potassium (K⁺)-selective, Shaker-type channels, particularly AKT1, in primary K⁺ acquisition in roots of higher plants has long been of interest, particularly in the context of low-affinity K⁺ uptake, at high K⁺ concentrations, as well as uptake from low-K⁺ media under ammonium (NH₄⁺) stress. We recently demonstrated that K⁺ channels cannot mediate K⁺ acquisition in roots of intact barley (Hordeum vulgare L.) seedlings at low (22.5 µM) external K⁺ concentrations ([K⁺]_ext) and in the presence of high (10 mM) external NH₄⁺, while the model species Arabidopsis thaliana L. utilizes channels under comparable conditions. However, when external NH₄⁺ was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K⁺ influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K⁺ influx, illustrating a hitherto unexplored magnitude and rapidity of K⁺-uptake capacity and plasticity. Here, we expand on our previous work by offering further characterization of channel-mediated K⁺ fluxes in intact barley, with particular focus on anion effects, root respiration and pharmacological sensitivity and highlight key additions to the current model of K⁺ acquisition.     

The Os-AKT1 Channel Is Critical for K+ Uptake in Rice Roots and Is Modulated by the Rice CBL1-CIPK23 Complex

Potassium (K⁺) is one of the essential nutrient elements for plant growth and development. Plants absorb K⁺ ions from the environment via root cell K⁺ channels and/or transporters. In this study, the Shaker K⁺ channel Os-AKT1 was characterized for its function in K⁺ uptake in rice (Oryza sativa) roots, and its regulation by Os-CBL1 (Calcineurin B-Like protein1) and Os-CIPK23 (CBL-Interacting Protein Kinase23) was investigated. As an inward K⁺ channel, Os-AKT1 could carry out K⁺ uptake and rescue the low-K⁺-sensitive phenotype of Arabidopsis thaliana akt1 mutant plants. Rice Os-akt1 mutant plants showed decreased K⁺ uptake and displayed an obvious low-K⁺-sensitive phenotype. Disruption of Os-AKT1 significantly reduced the K⁺ content, which resulted in inhibition of plant growth and development. Similar to the AKT1 regulation in Arabidopsis, Os-CBL1 and Os-CIPK23 were identified as the upstream regulators of Os-AKT1 in rice. The Os-CBL1-Os-CIPK23 complex could enhance Os-AKT1-mediated K⁺ uptake. A phenotype test confirmed that Os-CIPK23 RNAi lines exhibited similar K⁺-deficient symptoms as the Os-akt1 mutant under low K⁺ conditions. These findings demonstrate that Os-AKT1-mediated K⁺ uptake in rice roots is modulated by the Os-CBL1-Os-CIPK23 complex.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Effects of deuterium oxide on growth, proton extrusion, potassium influx, and in vitro plasma membrane activities in maize root segments

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by ²H₂O in the incubation medium. Short-term exposure (30 min) to ²H₂O slightly reduced O₂ uptake and significantly increased ATP levels. ²H₂O inhibited H⁺ extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K⁺ (about 30 and 53%, respectively at 50% [v/v] ²H₂O). Experiments on plasma membrane vesicles showed that H⁺-pumping and ATPase activities were greatly inhibited by ²H₂O (about 35% at 50% [v/v] ²H₂O); NADH-ferricyanide reductase and 1,3-β-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by ²H₂O. Therefore, the effect of short incubation time and low concentration of ²H₂O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H⁺ -ATPase. K⁺ uptake was inhibited by ²H₂O only when K⁺ was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (E_m) was slightly hyperpolarized by ²H₂O at low K⁺, but was not affected at the higher K⁺ concentrations. These results suggest a relationship between H⁺ extrusion and K⁺ uptake at low K⁺ external concentration.     

Short-term experiments on ion transport by seedlings and excised roots : technique and validity

The absorption of K⁺ by excised roots of barley (Hordeum vulgare L. cv California Mariout) has been systematically compared with that of entire, undisturbed seedlings. Some experiments have also been done with wheat (Triticum aestivum L.) and an amphiploid obtained from a cross between it and salt-tolerant tall wheatgrass (Lophopyrum elongatum Host Löve [syn. Agropyron elongatum Host]). For all three genotypes, the rate of K⁺ absorption measured in a 20-min period was identical for entire 8-d-old seedlings and their excised roots within the experimental error. Manipulation gentler than root excision, viz. careful transfer of seedlings from one experimental solution to another, was also without effect on the rate of K⁺ absorption. Absorption of K⁺ measured by assay of its ⁸⁶Rb label in the tissue was identical with that measured by K⁺ depletion of the experimental solutions assayed chemically. For the plant materials and conditions of these experiments, the excised root technique for studying ion transport into roots is validated. The advantages of the technique, and findings differing from the present ones, are discussed.     

Lack of stimulation of renal (Na+ + K+)-ATPase by thyroid hormones in the rabbit

The effect of l-3,5,3′-triiodothyronine (T₃) and thyroxine (T₄) on (Na⁺ + K⁺)-ATPase activities was examined in rabbit kidneys because in this tissue almost 80% of the metabolism is connected to active sodium transport. T₃-receptor concentrations were estimated as 0.62 and 0.80 pmol/mg per DNA in the cortex and outer medulla, respectively. A dose of 0.5 mg T₃/kg body weight for 3 days increased basal metabolic rate by almost 60%, and the mitochondrial 1-α-glycerophosphate dehydrogenase activity was increased by 50% in both the cortex and medulla. (Na⁺ + K⁺)-ATPase activity in the liver was raised by almost 50%. However, no changes in (Na⁺ + K⁺)-ATPase activities or binding sites for [³H]ouabain in either the kidney cortex or medulla could be observed. T₄ at 16 mg/kg daily for 14 days was also without effect on renal (Na⁺ + K⁺)-ATPase activities. Furthermore, the response to T₃ was absent at high sodium excretion rates induced by unilateral nephrectomy and extracellular volume expansion. Thus, despite stimulation of basal metabolic rate and renal 1-α-glycerophosphate dehydrogenase activity by T₃ and T₄, the (Na⁺ + K⁺)-ATPase activity in the rabbit kidney is identical in euthyroid and hyperthyroid states. However, thyroid hormones prevent the normal natriuretic response to extracellular volume expansion.     

Calcium inhibition of potassium absorption in corn roots

Calcium (or magnesium) sulfate or chloride was found to inhibit energy dependent potassium transport in excised corn roots. This Ca²⁺ inhibition of K⁺ transport was most pronounced during the initial phases of transport. As the absorption periods were lengthened the effect of Ca²⁺ gradually changed from an inhibition to a typical promotion (after about 30-45 mins) of K⁺ transport. Kinetic analysis indicated the inhibition to be of a non-competitive nature.

Identical experiments with excised barley roots showed that CaSO₄ had no effect on K⁺ absorption whereas CaCl₂ had a typical stimulatory effect on K⁺ absorption. Kinetic analysis indicated that both corn and barley have efficient K⁺ transporting systems but barley roots are approximately 5 times more active (on a fr wt basis) than corn roots.

These results illustrate the hazards involved in applying results obtained with 1 (or even several) plant species to all species.

     

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K⁺ analogue, ⁸⁶Rb⁺, from prelabelled cells, and the influx of ²²Na⁺. The permeabilizing activity of PA was limited to a proteolytically activated form (PA_N) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PA_N facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PA_N permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.     

High affinity k uptake in maize roots: a lack of coupling with h efflux

We report here on the putative coupling between a high affinity K⁺ uptake system which operates at low external K⁺ concentrations (K_m = 10-20 micromolar), and H⁺ efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K⁺(⁸⁶Rb⁺) influx, and the simultaneous measurement of net K⁺ and H⁺ fluxes associated with individual cells at the root surface with K⁺- and H⁺-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K⁺ and H⁺ ions within the unstirred layer at the root surface. No evidence for coupling between K⁺ uptake and H⁺ efflux could be found based on: (a) extremely variable K⁺:H⁺ flux stoichiometries, with K⁺ uptake often well in excess of H⁺ efflux; (b) dramatic time-dependent variability in H⁺ extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K⁺ uptake system (to changes in external pH) when net K⁺ uptake, unidirectional K⁺(⁸⁶Rb⁺) influx, and K⁺-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K⁺ absorption into maize root cells is not mediated by a K⁺/H⁺ exchange mechanism. Instead, it is either due to the operation of a K⁺-H⁺ cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K⁺-ATPase as reported for Escherichia coli and Saccharomyces.     

Potassium transport in corn roots : I. Resolution of kinetics into a saturable and linear component

Influx isotherms were obtained for ⁸⁶Rb⁺ uptake into 2-cm corn (Zea mays [A632 × (C3640 × Oh43)] root segments for both low- (0.2 millimolar CaSO₄) and high-salt (0.2 millimolar CaSO₄ + 5 millimolar KCl) grown roots. Unlike the discontinuous curves usually presented for K⁺ influx, our isotherms were smooth, nonsaturating curves that approached linearity at K⁺ (Rb⁺) concentrations above 1 millimolar. The kinetics for K⁺ transport could be resolved into saturable and linear components. The saturable components yielded K_m values of 16 and 86 micromolar for low- and high-salt roots, respectively, while V_max values were 5.62 and 1.85 moles per gram fresh weight per hour. Results of experiments with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS) indicated that the saturable and linear components were independent mechanisms of K⁺ transport.

Short-term NEM exposures (30 seconds to 5 minutes) selectively inhibited the saturable system, but had little effect on the linear component. Increasing NEM exposures resulted in further inhibition and subsequent abolition of the saturable component; the linear component exhibited limited NEM sensitivity. PCMBS elicited the same general inhibitory trends, although it was less effective as a saturable component inhibitor.

The effects of NEM and PCMBS on K⁺ efflux were also studied. Short NEM exposures had no effect on cytoplasmic efflux, while inhibiting vacuolar efflux significantly. From these data, it is unclear at which site(s) NEM is acting. A more complex response was obtained with PCMBS, where a monophasic efflux curve was observed. Analysis indicated that the vacuolar efflux was stimulated, while the cytoplasmic component was abolished.

The nature of the linear component is discussed, and it is proposed that the mechanism may be more complex than simple facilitated diffusion.