The Fungal H+-Atpase from Neurospora Crassa Reconstituted

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1599-1603, February 1995 Biochemistry

The fungal H+-ATPase from Neurospora crassa reconstituted with fusicoccin receptors senses fusicoccin signal MAURO MARRA*, ALESSANDRO BALLIOtt, PATRIZIA BATTIROSSI*, VINCENZO FOGLIANO§, MARiA RosARIA FULLONEt, CLIFFORD L. SLAYMANI, AND PATRIZIA ADUCCI* *Department of Biology, University of Rome "Tor Vergata," Via 0. Raimondo, 00173 Rome, Italy; tDepartment of Biochemical Sciences "A. Rossi-Fanelli," University of Rome "La Sapienza," p.le Aldo Moro 5, 00185 Rome, Italy; §Department of Food Science, University of Naples "Federico II," Parco Gussone, 80155 Portici, Naples, Italy; and 1Department of Cellular and Molecular Physiology, Yale University, 333 Cedar Street, New Haven, CT 06510-8026 Communicated by Winslow R Briggs, Carnegie Institution of Washington, Stanford, CA, November 7, 1994

ABSTRACT Fusicoccin affects several physiological pro- MATERIALS AND METHODS cesses regulated by the plasma membrane H+-ATPase in higher plants while other organisms having P-type H+- Chemicals. FC was prepared as described by Ballio et al. (19). ATPases (e.g., fungi) are 9-Amino-6-chloro-2-methoxyacridine (ACMA) and oxonol V fusicoccin-insensitive. We have pre- were purchased from Sigma and from Molecular Probes, respec- viously shown that fusicoccin binding to its receptor is nec- tively. Analytical grade chemicals were used throughout. essary for H+-ATPase stimulation and have achieved the Plant Material. Maize seeds (Zea mays L., var Logos) from functional reconstitution into liposomes of fusicoccin recep- Italian Dekalb (Mestre) were germinated and grown for 7 days tors and the H+-ATPase from maize. In this paper we show as reported (10). that fusicoccin sensitivity can be conferred on the H+-ATPase N. crassa H+-ATPase. The enzyme was purified by the from Neurospora crassa, a fungus insensitive to fusicoccin. In procedure of Bowman et al. (20). The concentration of the fact, H+ pumping by purified H-+ATPase from Neurospora H+-ATPase in the pooled purified fractions was 2.8 mg of crassa reconstituted into liposomes containing crude or par- protein per ml, with a specific activity of 20.5 ,tmol of Pi per tially purified fusicoccin receptors from maize was markedly min per mg of protein. The enzyme was diluted 1:15 for enhanced by fusicoccin. The stimulation of H+ pumping by reconstitution experiments. fusicoccin is dependent upon pH, fusicoccin, and protein FC Receptors from Maize. Crude solubilized FC receptors concentration, as was reported for the system reconstituted were prepared from maize shoots as described (21). They were with both proteins from maize. partially purified by the procedure of Aducci et al. (22), omit- ting the preincubation with labeled FC. After elution from the Binding of the fungal toxin fusicoccin (FC) (1, 2) to specific Bio-Gel hydroxyapatite column (Bio-Rad) in 150 mM sodium receptors (3, 4) in plant cells elicits a variety of effects that are phosphate (pH 6.7), all protein-containing fractions were ultimately dependent on the stimulation of the plasma mem- pooled, concentrated, and fractionated by HPLC on the an- brane H+-ATPase (5). Although FC stimulation of HI pump- ionic exchanger. Receptor-containing fractions were detected ing and phosphohydrolytic activity ofthe H+-ATPase has been by [3H]FC binding and used in reconstitution experiments as shown in different plant systems (6-10), the nature of the such or after concentration. functional relationship between FC receptors and the H+- Reconstitution Experiments. Liposomes were prepared as ATPase is still unclear. described (10) with minor modifications. A mixture of 25 mg In the last few years, we have developed (8, 10) an experi- of soybean phosphatidylcholine (type II, Sigma) and 25 mg of mental approach for the study of FC signaling based on sheep brain phosphatidylethanolamine (type II, Sigma) was simultaneously incorporating FC receptors and H+-ATPase sonicated to optical clarity in 1 ml of 10 mM Tris-Mes, pH 6.7/1 from maize into liposomes. The results have clearly demon- mM dithiothreitol/1 mM EDTA/100 mM KCl. Insertion into strated that binding of FC to its receptor is a prerequisite for liposomes of the purified ATPase and crude or partially pu- H+-ATPase stimulation. rified FC receptors was achieved as follows: ATPase [2 ,tg (10 Whereas all plant species tested so far, from mosses to ,ul)] was added to 3 ,ul of liposomes and 3 ,ul of 200 mM octyl angiosperms (11, 12), are sensitive to FC and contain FC glucoside. Aftervigorous mixing, the mixture was frozen for 10 receptors (3, 4, 9, 11-17), all other organisms investigated are min at -70°C and thawed at room temperature. Finally, 4 ,ul insensitive (16 jig of protein) of crude receptors or 10 ,ul (20 jig of to FC (12, 18) and appear devoid of FC-binding protein) of partially purified receptors was added, the freeze- capability (12). The occurrence in yeasts and fungi of plasma thaw cycle was repeated once more, and the mixture was added membrane H+-ATPases structurally and functionally related to 3 ml of assay buffer, to dilute octyl glucoside below its to higher plant ATPases raises the possibility that insensitivity critical micellar concentration. to FC might be due to the absence of an FC perceptor system. Proton Transport Assay. HI translocation into liposomes Our technique of reconstituting the components of the FC was followed by monitoring the fluorescence quenching of signaling pathway into liposomes appeared suitable for testing ACMA by means of a Perkin-Elmer model LS50 spectrofluo- this hypothesis. rimeter, at excitation and emission wavelengths of 430 and 500 The present work reports the reconstitution of FC receptors nm, respectively. Experiments were carried out as described from maize with the H+-ATPase from Neurospora crassa in (10), except that the quenching buffer was at pH 6.7. The initial liposomes and the demonstration that the HI pumping of this rate of quenching was expressed as the percentage of total system is stimulated by FC. fluorescence per min per mg of protein. The value obtained

The publication costs of this article were defrayed in part by page charge Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine; FC, fu- payment. This article must therefore be hereby marked "advertisement" in sicoccin. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 1599 Downloaded by guest on September 25, 2021 1600 Biochemistry: Marra et al. Proc. Natl. Acad Sci USA 92 (1995) after addition of ammonium sulfate was taken as 100% fluo- 6 rescence. Calibration of ApH. Fluorescence changes of ACMA were related to intravesicular pH changes by plotting the ratio of the intravesicular and extravesicular concentrations of the probe as a function of imposed pH gradients (23, 24). Liposomes were reconstituted in 200 mM Tris-Mes at pH values from 4.5 4 4. to 6.0. Aliquots (3 pl) were diluted in 3 ml of 10 mM Tris-Mes, pH 6.5/190 mM NaCl/1 ,uM ACMA and fluorescence quench- 3-6 ing was monitored. At the end of the experiment, the pH gradient was collapsed by adding 30 ,lI of 1 M (NH4)2SO4, and the resulting fluorescence value was taken as 100%. The molarity of ACMA inside and outside the liposomes was 2- calculated from the fluorescence quenching on the basis of the 0 1 2 3 measured internal volume of vesicles. This value was obtained ApH by determining the amount of 5(6)-carboxyfluorescein entrapped by liposomes during reconstitution as described by FIG. 2. Distribution of ACMA between intravesicular space and Goormaghtigh and Scarborough (25). external solution as a function of imposed pH gradient. Proteolipo- Membrane Potential Assay. MgATP-dependent hyperpo- somes reconstituted at pH 4.5 to 6.0 were diluted into reaction medium at pH 6.5. The percentage quenchingwas used to calculate the molarity larization of proteoliposomes was monitored as the fluores- of ACMA inside and outside the vesicles by using the measured cence quenching of oxonol V, by means of a Perkin-Elmer intravesicular volume (0.4 ,ul/mg of phospholipid). LS50 spectrofluorimeter at excitation and emission wavelengths of580 and 640 nm, respectively. The composition of the after the first freeze-thaw cycle. As shown in Fig. 1, where a quenching buffer was as follows: 10 mM Hepes-KOH (pH 6.7), typical experiment is reported, a short preincubation of pro- 100 mM potassium acetate, 5 mM (NH4)2SO4, and 4 ,uM teoliposomes with micromolar concentrations of FC caused, oxonol V. Samples of Neurospora ATPase were reconstituted upon energization by ATP, a marked increase over the control in the conditions described above and tested in the presence or of the initial rate of HI pumping and of the steady-state level absence of FC; the reaction was started by adding 12 pl of 1 of the HI difference. The extent of FC stimulation was vari- M MgSO4. The total volume was 3 ml. Vanadate at 150 ,uM was able, between 2- and 5-fold of the initial rate of proton pump- added at the stationary level of quenching to block the pump and to monitor the kinetics of the fluorescence increase. ing. The magnitude of the pH gradient was estimated by using Analytical Methods. Protein concentrations were estimated the calibration curve reported in Fig. 2. An internal volume of by the method of Bradford (26). ATPase activity was measured 0.4 ,ul/mg of phospholipids was calculated for our liposomes, as described by Serrano (27). a value consistent with those reported for similar vesicles (23). Under optimal conditions an extra acidification of 0.4 pH unit over the control was obtained upon addition of FC. As observed RESULTS AND DISCUSSION in the homologous system (8, 10), a period of at least 6 min of FC receptors from maize shoots and the plasma membrane preincubation with FC was necessary for efficient stimulation; no H+-ATPase from N. crassa have been incorporated into phos- effect was detected upon addition of FC and ATP together. phatidylcholine/phosphatidylethanolamine liposomes. These The stimulation of HI pumping was dependent on FC proteoliposomes were quite active in HI translocation, and concentration (Fig. 3) in a way similar to that observed with the interestingly, H+ pumping was stimulated by FC. As reported homologous system (10): maximal rates occurred at 1 p.M FC with maize H+-ATPase (10), the best conditions for reconsti- but rates decreased at higher FC concentrations. tution were obtained by a combination of freeze-thaw and To ascertain whether this could be due to a concurrent FC detergent dilution procedures and by adding FC receptors effect on the lipid bilayer, experiments to evaluate the passive permeability of proteoliposomes to protons in presence of

100 12,000 -

- 80 ? E 2~~~ -R %6i1\ > _ 8,000-

e,- 4,0

0 5 10 15 20 Time, min 0 10-8 10-7 10-6 10-5 10-4 FIG. 1. Effect of FC on ATP-driven HI pumping in proteoliposomes FC, M reconstituted with N. crassa H+-ATPase and crude FC receptors from maize. Acidification of proteoliposomes was measured by the quenching FIG. 3. FC-concentration-dependent stimulation of HI pumping. of ACMA fluorescence after addition of ATP (solid arrow). Quenching Amounts of H+-ATPase and crude FC receptors were the same in all was reversed by addition of (NH4)2SO4 (open arrow). Solid line, proteo- samples (2 ,ug and 16 ,ug, respectively). Samples were assayed in liposomes containing H+-ATPase from N. crassa and FC binding sites duplicate; variations were <10%. The decrease of stimulation by FC from maize (control); dashed line, same proteoliposomes but incubated concentrations >10-6 M is due to increased passive permeability of 6 min with 1 ,uM FC before ATP addition. proteoliposomes to protons caused by FC. Downloaded by guest on September 25, 2021 Biochemistry: Marra et al. Proc. NatL Acad Sci. USA 92 (1995) 1601 various FC concentrations were carried out by the method of Szponarsky et al. (28). As shown in Fig. 4 Upper, in presence of 50 ,uM FC, the H+ pumping of the ATPase and the relaxation time of the proton gradient were markedly reduced. bi These effects became negligible for toxin concentrations below 4

10 ,uM. The overall data suggest that high concentrations . (>10-5 M) of FC increase the permeability of liposomes to .C 3000 protons and that this effect can mask the specific stimulation F0.~. of pumping brought about by interaction with its receptors. Similar results were obtained by investigating the effect of FC X =4 concentration on the passive permeability of proteoliposomes by 10.0 monitoring the membrane potential of proteoliposomes by oxo- cr 1000- nol V fluorescence quenching. As shown in Fig. 4 Lower, 50 JIM FC reduced the ATP-dependent potential hyperpolarization of proteoliposomes and speeded up loss ofoxonol V quenching after enzyme blockade at the stationary state. On the other hand, both 5.5 6 6.7 7.5 hyperpolarization and relaxation ofthe membrane potential were pH practically unaffected by FC levels as low as 1 ,uM. FIG. 5. pH dependence of FC stimulation of H+ pumping. Exper- The pH dependence of the FC effect is reported in Fig. 5: the imental conditions were as in Fig. 3. Bars: hatched, control proteoli- stimulation was optimal for pH 6.0-6.7 and was lost at higher or posomes; open, same proteoliposomes but incubated with 1 ,uM FC. lower pH values, known to affect negatively both FC binding to receptors (29) and H+-ATPase hydrolytic activity (30). Reconstitution experiments were carried out also with partially purified FC receptors. Fig. 6 reports an SDS/PAGE profile of these proteins showing that the 90-kDa and 30-kDa bands, identified as components of a putative FC receptor complex (17, 22, 31, 32), are prominent. The increase of their specific activity at this stage was 100-fold. The stimulatory effect of FC was markedly influenced by the amount of receptors entrapped in liposomes for both crude and purified proteins (Fig. 7). A similar stimulation was obtained with either crude or purified receptors. Purified pro- 2e teins were more effective than comparable amounts of the crude ones, but nevertheless, the increase did not parallel that 2 min of specific activity. This discrepancy might be explained by the partial loss during purification of an unknown component, necessary for FC signaling. Since Fig. 7 shows a declining rather than a saturating profile upon incorporation of increasing amounts of receptors, a nonspecific effect of increased proteoliposome leakiness was B A

C ,U,.; ._r B A

10 1 min W',p,M11--mw

U FIG. 4. Effect of FC concentration on the ATP-dependent ApH and At of proteoliposomes. (Upper) Traces refer to ACMA fluorescence quenching, after the addition of ATP (solid arrow). Traces (relaxation times are in parentheses): A, control (2.8 min); B, 1 ,uM FC (2.5 min); C, 50 ,LM FC (1.3 min). To follow the relaxation of the gradient, the pump was blocked by adding 22 ,l4 of 0.2 M vanadate (open arrow). (Lower) Traces refer to oxonol V fluorescence quench- FIG. 6. SDS/PAGE of partially purified FC receptors. Lanes: A, ing, after the addition of Mg2+ (solid arrow). The pump was blocked molecular mass standards from Bio-Rad (97.4, 66.2, 42.7, 31.0, and as in Upper. Traces (relaxation times are in parentheses): A, control 21.5 kDa); B, partially purified FC receptors. Arrows indicate putative (10.2 min); B, 1 ,uM FC (9.6 s); C, 50 ,uM (6.2 min). FC receptors with estimated molecular masses of 90, 33, and 31 kDa. Downloaded by guest on September 25, 2021 1602 Biochemistry: Marra et al. Proc. Natl. Acad ScL USA 92 (1995) It is known that H+-ATPases from fungi and plants are structurally related and data in favor of similar regulatory mechanisms are emerging. In fact, it has been shown that the WI C-terminal region of fungal (33) and plant (34) H+-ATPases is an autoinhibitory domain whose displacement leads to a fully activated enzyme. Despite the limited overall homology between fungal and plant H+-ATPases, it has been demon- strated that a peptide covering a region of the plant C terminus homologous to the yeast C terminus inhibits the activated trypsin-treated plant H+-ATPase (35). Moreover, recent papers (36-38) have shown that trypsin * treatment of plasmalemma inside-out vesicles results in a fully activated FC-insensitive H+-ATPase, thus suggesting that the C-terminal inhibitory domain of the enzyme is involved also in the mechanism of FC stimulation. In conclusion, our results favor a general role for FC re- 0 20 40 180 ceptors. The FC-FC receptor complex appears to activate the FC receptors, ,g of total protein H+-ATPase by interacting with the multifunctional C-terminal domain, which is conserved among P-type ATPases (34, 35). FIG. 7. Effect of the amount ofcrude and purified FC receptors on In light of these observations, the insensitivity of fungi to FC FC stimulation of HI pumping. All samples were incubated with 1 ,uM seems to be due uniquely to the absence of a FC perception/ FC and contained 2 ,ug of H+-ATPase and reconstituted proteins as transduction system that appears to have occurred later in their indicated. Solid circles, crude FC receptors; open circles, partially purified FC receptors. evolution (from mosses) and very likely acts in plants as a detector system for physiological ATPase effectors (39). likewise supposed. This hypothesis was tested by investigating the effect of increasing amounts of incorporated proteins on We dedicate this paper to Prof. E. Marre on the occasion of his 75th the passive permeability of proteoliposomes to protons. Fig. 8 birthday. This work was supported by the National Research Council of Italy, Special Project RAISA, Subproject No 2. Paper No. 1994. shows that the HI pumping of proteoliposomes reconstituted A.B. thanks the National Research Council of Italy, Special Project with H+-ATPase was progressively reduced upon incorpora- "Chimica Fine 2," Subproject No.3. The work was also supported by tion of increasing amounts ofcrude FC receptors. On the other the Italian Ministry of Agriculture and Forestry, by the Italian Min- hand, when the pump was blocked at the steady state, the rate istry of University and Scientific Research, and by a contract of the of gradient relaxation increased. Also in this case, data indi- Commission of European Communities (BRIDGE). cated that the decrease of FC stimulation was due to a nonspecific effect of increased permeability of proteoliposomes. 1. Ballio, A., Chain, E. B., De Leo, P., Erlanger, B. F., Mauri, M. & The results reported in the present study on the stimulatory Tonolo, A. (1964) Nature (London) 203, 297. 2. Ballio, A., Brufani, M., Casinovi, C. G., Cerrini, S., Fedeli, W., effect of FC on N. crassa H+-ATPase, mediated by FC recep- Pellicciari, R., Santurbano, B. & Vaciago, A. (1968) Experientia tors, suggest the occurrence of a conserved regulatory pathway 24, 631-635. for FC signaling in fungal and plant H+-ATPases. 3. Dohrmann, U., Hertel, R., Pesci, P., Cocucci, S. M., Marre, E., Randazzo, G. & Ballio, A. (1977) Plant Sci. Lett. 9, 291-299. 4. Ballio, A., Federico, R., Pessi, A. & Scalorbi, D. (1980) Plant Sci. ; ~~~~~DC Lett. 18, 39-44. 30 5. Marre, E. (1979) Annu. Rev. Plant Physiol. 30, 273-288. 6. Rasi-Caldogno, F. & Pugliarello, M. C. (1985) Biochem. Biophys. Res. Commun. 133, 280-285. 7. Rasi-Caldogno, F., De Michelis, M. I., Pugliarello, M. C. & Marre, E. (1986) Plant Physiol. 82, 121-125. 8. Aducci, P., Ballio, A., Blein, J-P., Fullone, M. R., Rossignol, M. & Scalla, R. (1988) Proc. Natl. Acad. Sci. USA 85, 7849-7851. 550 600 650 9. Schulz, S., Oelgem6ller, E. & Weiler, E. W. (1990) Planta 183, ?60 83-91. ~~~~~~~~DD 10. Marra, M., Ballio, A., Fullone, M. R. & Aducci, P. (1992) Plant Physiol. 98, 1029-1034. B 11. Graniti, A. (1964) Phytopathol. Medit. 3, 125-128. 12. Meyer, C., Waldkotter, K, Sprenger, A., Schlosser, U. G., Luther, M. & Weiler, E. (1993) Z. Naturforsch. 48c, 595-602. 13. Stout, R. G. & Cleland, R. E. (1980) Plant Physiol. 66, 353-359. 20 A 14. Pesci, P., Tognoli, L., Beffagna, N. & Marre E. (1979) Plant Sci. Leut. 15, 313-322. 15. De Michelis, M. I., Pugliarello, M. C. & Rasi-Caldogno, F. (1989) Plant Physiol. 90, 133-139. 200 400 600 800 1000 16. Meyer, C., Fayerabend, M. & Weiler, E. W. (1989) Plant Physiol. Time, s 89, 692-696. 17. Oecking, C. & Weiler, E. W. (1991) Eur. J. Biochem 199, 685-689. FIG. 8. Effect of the amount of crude receptors on proton per- 18. Chain, E. B., Mantle, P. G. & Milborrow, B. W. (1971) Physiol. meability of proteoliposomes. Traces refer to ACMA fluorescence Plant Pathol. 1, 495-514. quenching, after the addition ofATP (solid arrow). Samples contained 19. Ballio, A., Carilli, A., Santurbano, B. & Tuttobello, L. (1968) the same amount (2 ,ug) of H+-ATPase and increasing amounts of Ann. lst. Super. Sanit& 4, 317-332. crude FC receptors, as follows. Traces: A, control; B, 16 AL&g; C, 32 L&g; 20. Bowman, B. J., Blasco, F. & Slayman, C. W. (1981)J. Biol. Chem. D, 64 ,ug. To follow the relaxation of the gradient, the pump was 256, 12343-12349. blocked by adding 30 ,ul of 0.5 M EDTA (open arrow). (Inset) En- 21. Aducci, P., Ballio, A. & Federico, R. (1982) in Plasmalemma and largement of traces after the enzyme blockade is shown to highlight the Tonoplast, Their Function in the Plant Cell, eds. Marme, D., different slopes of relaxation curves. Marre, E. & Hertel, R. (Elsevier, Amsterdam), pp 279-284. Downloaded by guest on September 25, 2021 Biochemistry: Marra et al. Proc. NatL Acad Sci USA 92 (1995) 1603

22. Aducci, P., Ballio, A., Fogliano, V., Fullone, M. R., Marra, M. & 31. de Boer, A., Watson, B. A. & Cleland, R. E. (1989) PlantPhysiol. Proietti, N. (1993) Eur. J. Biochem. 214, 339-345. 89, 250-259. 23. Perlin, D. S., San Francisco, M. J. D., Slayman, C. W. & Rosen, 32. Feyerabend, M. & Weiler, E. W. (1989) Planta 178, 282-290. B. P. (1986) Arch. Biochem. Biophys. 248, 53-61. 33. Portillo, F., Eraso, P. & Serrano, R. (1991) FEBSLett. 287,71-74. 24. Brauer, D., Tu, S. I., Hsu, A. F. & Thomas, C. E. (1989) Plant 34. Palmgren, G. M., Sommarin, M., Serrano, R. & Larsson, C. Physiol. 89, 464-471. (1991) J. Biol. Chem. 266, 20470-20475. 25. Goormaghtigh, E. & Scarborough, G. A. (1986) Anal. Biochem. 35. Serrano, R., Portillo, F., Monk, B. & Palmgren, G. M. (1992)Acta 159, 122-131. Physiol. Scand. 146, 131-146. 26. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 36. Johansson, F., Sommarin, M. & Larsson, C. (1993) Plant Cell 5, 27. Serrano, R. (1989) Methods Enzymol. 157, 533-544. 321-327. 28. Szponarsky, W., Wansuyt, G. & Rossignol, M. (1991) Phytochem- 37. Rasi Caldogno, F., Pugliarello, M. C., Olivari, C. & De Michelis, istiy 30, 1391-1395. M. I. (1993) Plant Physiol. 103, 391-396. 29. Aducci, P., Fullone, M. R. & Ballio, A. (1989) Plant Physiol. 92, 38. Lanfermeijer, F. C. & Prins, H. B. (1994) Plant Physiol. 104, 1402-1406. 1277-1285. 30. Bowman, B. J., Blasco, F. & Slayman, C. (1977) J. Biol. Chem. 39. Aducci, P., Crosetti, G., Federico, R. & Ballio, A. (1980) Planta 252, 3357-3363. 148, 208-210. Downloaded by guest on September 25, 2021