H+ Antiport in Isolated Tonoplast Vesicles from - NCBI

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Nov 4, 1985 - ABSTRACT. Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Betavulgaris L.) were used to study the kinetics of a .... and/or pH changes by subtraction of rates obtained by addition of TMA+ .... values were changed in parallel, and a pH gradient of 2 units. 0. 20. 40. 60.
Plant Physiol. (1986) 80, 727-731 0032-0889/86/80/0727/05/$0 1.00/0

Kinetics of Ca2' /H+ Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L.1 Received for publication September 13, 1985 and in revised form November 4, 1985

EDUARDO BLUMWALD* AND RONALD J. POOLE

Plant Molecular Biology Center, Department of Biology, McGill University, Montreal, Quebec, Canada H3A IBI ABSTRACT Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were used to study the kinetics of a Ca2`lH antiport across this membrane. Ca2-dependent H' fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ApH-dependent Ca2" influx was measured radiometrically. Both Ho efflux and Ca2" influx displayed saturation kinetics and an identical dependence on external calcium with apparent K. values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg2+ but was inhibited by La3+ > Mn2+ > Cd2+. The apparent K. for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested.

The widespread presence of calmodulin in plant tissues, and the occurrence of enzymes regulated by Ca2" and calmodulin (9) suggest that the Ca2" concentration in the cytosol of plant cells, as in animals, plays an important role in the regulation of cell activities. The concentration of free Ca2" in the cytosol of Nitella and Chara was estimated by the injection of aequorin (24) to be in the range I0-` to 10-6 M, compared with >l0I M in the vacuole. The distribution of Ca2" in plant tissues (21) suggests that Ca2+ is actively transported out of the cytosol at the plasma membrane and tonoplast and is also accumulated by mitochondria and chloroplasts. Active transport of Ca2+ is also indicated by compartmental analysis of isotope fluxes (13). Studies of Ca2+ transport in isolated membrane fractions also indicate multiple transport sites. Ca2+ transport systems dependent on ATP but insensitive to proton conductors have been observed in microsomal membranes identified as plasma membrane (8) and ER (5, 6, 10). At least in the case of the putative plasma membrane fractions from higher plants, Ca2+ transport appears to be mediated by a Ca-ATPase, activated by calmodulin (7). In Neurospora, however, Ca2' efflux at the plasma membrane is achieved by Ca2+/H' antiport (23). Evidence for a Ca2+/H' antiport has been found in the tonoplast of carrot cells (6), Hevea latex (14), and yeast (17). Ca2"/ H+ antiport activity observed in incompletely identified membrane fractions from various tissues (11, 20, 22, 25) is probably also attributable to tonoplast vesicles. This Ca2+/H' antiport has not been characterized in detail, especially in higher plants, but it is clear that when experiments are designed so that the rate of Ca2+/H' antiport is not limited by the rate of H+-ATPase, the ' Supported by the Natural Sciences and Engineering Research Council of Canada and the Department of Education of Quebec.

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antiport can be very rapid (11). The present study characterizes the kinetics of Ca2+/H' antiport in an identified tonoplast vesicle fraction, in order to begin to assess its role in the control of Ca2" and H+ levels in cytoplasm and vacuole. MATERIALS AND METHODS

Plant Material. Fresh red beet (Beta vulgaris L.) with leaves intact were purchased commercially, stored at 4°C, and used within 1 week of purchase. Isolation of Tonoplast Vesicles. Vesicles identified as tonoplast were isolated as described before (2, 18), except that the homogenization medium contained 5 mM EDTA. The vesicles were preloaded with a buffer of desired ionic composition by suspension and sedimentation at 100,OOOg for 30 min. Final membrane pellets were resuspended in the same loading medium to 6 mg protein/ml and incubated at 40C for 2 h. Fluorescence Assays. The fluorescence quenching of acridine orange was used to monitor the formation and dissipation of inside-acid pH gradients (3, 12) across the membranes of the tonoplast vesicles. In all experiments, tonoplast vesicles (80 ,g protein) were added to 2 ml of buffer containing 5 gM acridine orange at 25°C. Subsequent changes in the fluorescence were monitored with a Perkin-Elmer spectrofluorimeter model LS-5 at excitation and emission wavelengths of 495 and 540 nm, respectively, and a slit width of 5 nm for both excitation and emission. During the measurement, the samples were continuously stirred. A present ApH was obtained by mixing tonoplast vesicles (pH, 6.0) with a buffer solution at pHo 8.0 (pH jump). These pH values were varied for the experiment of Figure 5. Vesicles were added and the dissipation of the initial quenching was recorded. Aliquots from stocks of 10 mM CaCl2 or TMA2CI were injected during continuous fluorescence recording and the resulting initial rates of fluorescence recovery were determined. To prevent pH changes in the outside buffer, all stock solutions were buffered at pH 8.0. The initial rates were determined by drawing the tangents of the recorded traces obtained in the first 10 s following the addition of salts. The initial rates ofdissipation of the pH gradient by Ca2+ were corrected for solution volume and/or pH changes by subtraction of rates obtained by addition of TMA+ and were expressed as rate of change in fluorescence per second. ApH Dependent Ca2` Uptake. Tonoplast vesicles (50 yg protein) loaded with buffer at pH 6.0 were resuspended in a total volume of 0.5 ml containing 25 mM Tris/Mes (pH 8.0), 10 mM K-gluconate, 250 mm sorbitol, and 5 ,uM valinomycin. After 30 s, Ca2+ uptake was started by adding aliquots of 1 mm CaCl2 plus 45CaC12 (0.018 uCi/,ug). After 10 s of incubation the vesicles were collected on 0.45 um Millipore filters and washed with ice cold

2Abbreviations: TMA, tetramethylammonium.

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BLUMWALD AND POOLE

buffer containing 25 mm Tris/Mes (pH 8.0) and 250 mM sorbitol. The entire process from the addition of 45Ca2" until the termination of the uptake assay by washing and filtration was carried out in 15 s. The correction for extravesicular 45Ca2" was carried out by adding 3H-inulin (0.01 gCi/,ug) to the uptake medium. The radioactivity retained on the filter was determined in 10 ml Aquasol solution in a liquid scintillation counter. The term ApH-dependent Ca2+ uptake is defined as Ca2+ uptake in the presence of a pH gradient (induced by the "pH jump") minus the uptake of Ca2+ with membranes previously incubated with 1 mm NH4Ci, which collapsed the ApH across the tonoplast vesicles. Ca2+ uptake is defined as the amount of Ca2+ taken up per mg protein after 15 s of addition of Ca2+. Nonspecific Ca2+ uptake was about 10% of the maximum value of ApH-dependent Ca2+ uptake. Protein Determinations. Protein was measured by a modification of the dye-binding method (4) as previously described (3). Chemicals. Acridine orange was purchased from Sigma Chemical Co., 45CaCl2 and Aquasol from New England Nuclear, and [3H]Inulin from Amersham. All chemicals were the highest analytical grade commercially available.

Plant Physiol. Vol. 80, 1986

generated by pH jumps in the presence of equimolar K+ concentrations (10 mM) across the membrane and 5 AM valinomycin, which abolished the electrical membrane potential across the tonoplast membranes (2, 3). In the experimental range, the initial rate of H+/Ca2` exchange (fluorescence recovery), in the presence of K+ plus valinomycin displayed saturation kinetics with respect to extravesicular Ca2+ concentrations (Fig. 2). An Eadie-Hofstee plot for the kinetic data yielded a straight line with an apparent Km of 43.9 ,uM (Fig. 2, inset). ApH Dependent Ca2` Uptake. The pH gradient generated by pH jumps was used to drive 45Ca2+ accumulation in the tonoplast vesicles. Mg2", present in the medium, served to eliminate a significant amount of nonspecific Ca2+ binding to the membranes, without a significant effect on the ApH generated by pH jumps (see below). The ApH-dependent 45Ca2e accumulation by tonoplast vesicles was very fast, linear for the first 10 s, and reached a maximum after 20 s, following the addition of 45Ca2+ to the extravesicular medium (Fig. 3). A comparison of the effect of different cations on ApH dependent Ca2` accumulation is presented in Table I. La'+, which has been shown to displace Ca2+ from its binding sites and to inhibit Ca2+ transport in various tissues (15), inhibited Ca2+

RESULTS 2.0 Ca2" Dependent H' Efflux. The effect of Ca2+ on the dissipation of a transmembrane pH was tested in tonoplast vesicles. 1.5 The addition of vesicles equilibrated at pH 6.0 to a pH 8.0 buffer (pH jump) caused quenching of acridine orange fluorescence (Fig. 1). It has previously been shown that the quenching of the 0coUfluorescent signal is caused by the accumulation of the fluores1.0 cent dye in the intravesicular space (3, 12) and that the rate of change of quenching of acridine dyes is proportional to the flux of protons (1). After an initial high degree of quenching and 0.5 partial recovery (