proton antiport systems in Escherichiacoli

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Cation/Proton Antiport Systems in Escheriehia coli Robert N. Brey, Jeanne C. Beck and Barry P. Rosen Department of Biological Chemistry University of Maryland School of Medicine Baltimore, Maryland 21201 Received

July

3,1978

SUMMARY: Three distinct systems which function as proton/cation antiports have been identified in E. coli by the ability of the ions to dissipate the ApH component of the protonmotive forceinverted vesicles. System I exchanges H+ for K+, Rb+ or Na+; System II has Na+ and Li+ as substrates; and System III catalyzes proton exchange for Ca2+, Mn2+ or Sr 2+. INTRODUCTION: Transport systems which derive their energy from previously-formed electrochemical gradients utilize three basic mechanismsfor energy coupling, as defined by Mitchell (1,2): symports, uniports and antiports.

To date there have been few data

available on the number and types of antiport systems which exist in bacteria. West and Mitchell (3) first postulated the existence of a Na+/H+ antiporter in AE coli based on the effect of Na+ on proton fluxes in anaerobic cells. Shuldiner and Pishkes(4), investigating the effect of Na+ on the pH gradient produced by everted vesicles, demonstrated that Na+ and Li+ share a common system. Na+/H+ antiports have been demonstrated in Salmonella typhimurium (5), Halobacterium halobium (6), Azotobacter vinelandii (7) and Alteromonas haloplanktis (8) and are probably universally distributed among bacteria. Another antiport system found in many bacteria is the Ca2+/H+ antiport, first described in this laboratory (9) and later found in other bacteria (10,ll). exists in 5 halobium (12).

An analogousCa2i/Na’ antiport

In this communication we report the determination of the number and specifities of cation/proton antiporters found in everted membrane vesicles of -E. coli. Three systems exist, two of which exchange protons for monovalent cations and the third of the

which functions with divalent cations. The systems are 1) the Potassium/Proton Antiporter (KHA) whose substrates are K+, Rb+ and Na+; 2) the Sodium/Proton Antiporter (NHA) with substrates Na+ and Li+; and 3) the Calcium/Proton Antiporter (CHA), which transports Ca2+- Mn2+ or Sr2+ in exchange for H+. MATERIALS AND METHODS: E. coli K12 strain 7 (13) was grown to stationary phase -in a basal salts medium (14) supplemented with 54 mM glycerol as carbon source. Cultures were harvested, washed once and resuspendedto 5 volumes/gram of wet cells in a buffer consisting of 18 mM Tris-HCl, pH 7.2, containing 140 mM choline chloride, 0.5 mM dithiothreitol and 10% (v/v) glycerol (TCDG buffer). Eve&ted membrane vesicles were prepared by lysis of cells in a French press at 4,000 psi at 0 . The suspensionwas centrifuged for 10 min at 27,000 xg, and the pellet of unbroken cells was resuspendedin the original volume of TCDG buffer, put through the French press again, and recentrifuged. The two supernatant solutions were combined and centrifuged at 100,000 xg for 1 hr. The pelleted 0006-291x/78/0834-1588$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Fig. 1: Effect of cations on ApH. The pH gradient was estimated from the lactatedependent quenching of the fluorescence of quinacrine by everted membranevesicles, as described under Methods. Quenching was initiated by the addition of Tris D,L-lactate to 10 mM, final concentration. At the indicate$ times+either KCl, NaC,J+orCaCl was added to yield a final concentration of 5 mM for K or Na or 1 mM for Ca . A: Al? three salts were added prior to addition of Tris lactate; B: The cations were added sequentially following formation of ApH; C: Control.

membrane vesicles were washedonce and resuspendedto 2 to 5 mg of protein/ml in TCDG buffer. For storage vesicle suspensionswere mixed with an equal volume of pure glycerol and kept at -20’. The hpH was estimated from the energy-dependent quenching of quinacrine fluorescence, as described previously (15). The assay medium consisted of 10 mM Tris-HCl, pH 8, containing 140 mM choline chloride and 1 nM quinacrine-HCl in a final volume of 2 ml. Quenching was initiated by the addition of 10 ~1 of 2M D,L-lactate adjusted to pH 8 with Tris base. Each assay contained approximately 40 ug of membrane protein. Fluorescence was measuredwith an Aminco-Bowman Spectrofluorometer, with excitation at 420 nm and emission at 500 nm. Protein concentrations were determined by the method of Bradford (16) using bovine serum globulin as a standard. RESULTS: In a Tris-choline buffer oxidation of lactate produced approximately a 60% quenching of the fluorescence of quinacrine, reflecting the formation of a transmembrane pH gradient, acid interior, by everted vesicles (Fig. 1). It was found that three groups of compoundscould cause partial dissipation of ApH as detected by a decrease in quenching. These groups are represented in Fig. 1, curve B by the sequential addition of KCl, NaCl

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lmin

Ca

ib

Fig. 2: Specificity of the Potassium/Proton Antiporter. See Fig. 1 for details. RbCl was added to 5 mM, final concentration. Concentrations of other ions were as given in Fig. 1.

and CaC12. Addition of all three prior to addition of lactate (curve A) produced a level of quenching equivalent that found after sequential addition. In the concentrations used none of the ions had an effect on the oxidation of lactate. The specificity of the systems was determined both by the ability of ions to dissipate ApH and their ability to prevent other ions from doing so. Thus, RbCl was found to dissipate ApH in the same way that K+, Na+ or Ca2+ could (Pig. 2, curve C!). If KC1 were added before Rb+, no further effect of Rb+ on A pH was observed (Fig. 2, curve B). Likewise, addition of Rb+ prior to the addition of lactate resulted in the formation of a smaller ApH, and subsequent addition of K+ was without effect (Fig. 2, curve Al. However, Na+ and Ca2+ were both capable of dissipating A pH even in the presence of K+ and/or Rb+ (Fig. 2). Thus, it seemslikely that there is an antiport which exchanges H+ for either K+ or Rb+. While Ca2+ IS ’ not a substrate, it does appear that the system also uses Na+, since addition of Na+ prior to K+ or Rb+ prevented either of those from further

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Li ?

Li

t

Li

Fig. 3: Specificity of the Sodium/Proton Antiporter. LiCl were added to 10 mM, final concentration.

See Fig. 1 for details. NaCl and

dissipating ApH (data not shown). This system has been termed the Potassium/Proton Antiporter (KHA) system to distinguish it from what is most likely the major Na+/H+ antiport system, termed NHA, As shown in Fig. 3A, Li+ could dissipate ApH (curve ‘21,or, if added before the lactate, resulted in the formation of a smaller ApH (curve 1). In either ease, Na+ had no further effect on ApH (Fig. 3A). In the reciprocal experiments (Fig. 3B), Na+ inhibited the exchange of Li+ for H+. This defines the NHA system as Na+ or Li+ antiporter with H+. Preliminary data suggestthat neither K+ nor Rb+ are substrates of the NHA System. Finally no monovalent cations inhibited dissipation of the ApH gradient by Ca2+, nor did Ca2+ prevent monovalent cation/H+ exchange. As shown in Fig. 4A,Mn2+shares a common antiporter with Ca2+ in that Mn2+ dissipated the pH gradient in a reaction inhibited by Ca2+ and Mn2+ *m turn prevented the dissipation of ApH by Ca2i but not Nat. Similar results were obtained with Sr2t (Fig. 4B), but Mg2+, Ba2+, Co2+, and ruthenium red neither dissipated ApH nor prevented the dissipation by Ca2t (data not shown). Thus the Calcium/Proton Antiporter @HA) exchanges prOtOnSfor Ca2+, Mn2+, and Sr2+. In addition, La3+ could prevent the dissipation of ApH by Ca 2+ but alone appeared to increase ApH rather than decrease it. Moreover, at the concentrations of La3t used (20-

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Specificity of the Calcium/Proton Antiporter. See Fig. 1 for details. MnCl and Fig. 4: SrCl were added to 1 mM, final concentration. Concentrations of other ions wePe as gived in Fig. 1.

50 n M) there was a 1545% inhibition of lactate oxidation.

It is possible that La3+ is a

substrate of the CHA system but also acts as a permeant cation, increasing the magnitude of the pH gradient by dissipating A$ . We have often observed that A$ limits the size of A pH (unpublishedresults). DISCUSSION: A large number of active transport systems exist for the accumulation of solutes by cells. The function of many is clear, since metabolism and transport are intimately related. Neither the number nor function of efflux systems in bacterial cells is known. We have attempted here to define those ion transport systems which function as cation/proton antiports in everted vesicles of aE. coli. There are at least three; the KHA system for K+, Rb+ and Na+, the NHA system for Na+ and Li+, and the CHA system for Ca2+, Mn2+ and Sr2+. At present the function of these systems is unknown. We have speculated in the past that Ca2+ fluxes are involved in ehemotaxis (91, and a recent report by Ordal(17) suggeststhat such might be the case in B. -- subtilis. The NHA system could be involved in generation of electrochemical sodium gradients, necessary for energization of Na+/solute

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symports, such as that for glutamate in -E. coli (2,18). However, there is no evidence that sodiumorcalcium are either required for growth of cells or are deleterious to growth under most physiological conditions. Potassium, on the other hand, is absolutely required for growth, and there are a variety of active transport systems for the accumulation of K+ (19). What, then, is the function of a system which, presumably, would be responsiblefor extrusion of K+ from intact cells ? One likely possibility is for regulation of intracellular PH. In formation of a protonmotive force, there must be a limit to the change in pH which is tolerable to the cell. In fact, the intracellular pH is finely regulated, keeping near neutral under wide range of extracellular pH (20). This could not be accomplished simply by re-uptake of H+, since that would dissipateA$ as well. Exchange of H+ for another cation would allow a transfer of the energy of the pH gradient to another gradient, in this case, K+. It is possiblethat Na+ or Ca2+ could serve this purposeunder some conditions, but neither are present consistently nor required for growth as is K+. There must be a continuous high intercellular concentration of the ion to allow for large movements of Ht. Only Kt consistently satisfies that requirement, since it is continually accumulated to high concentrations. If both Kt efflux and influx occur simultaneously, why is there not a futile cycle, resulting in uncoupling? The most likely explanation is that the rate of efflux is low relative to influx. In the experiments described in this report complete dissipation of A pH does not occur with any of the ions. If cation/H’ exchanging ionophores are added (nigericin for K+, monensin for Nat, or A23187 for Ca2’), complete dissipation does happen. With the ionophores the rate of exchange must be faster than the rate of proton influx catalyzed by lactate oxidation. In the case of the endogenousantiporters, complete dissipation of ApH is prevented probably becausethe rate of exchange is slower than the rate of proton uptake. If Kt influx under physiological conditions is rapid enough, the Kt current should not uncouple the system. Thus, the system is not in thermodynamic equilibrium, but is in a steady state which is a function of the kinetics of the components. It is necessary to exercise caution in deriving thermodynamic parameters from techniques which measure steady states, such as quenching of quinacrine fluorescence or flow dialysis. Finally, it is apparent from this study that the gradients of Kt and Nat, if not Lit and Ca2’, must be considered in studies of bioenergetics. Since many studies utilize vesicle preparations in which Kt is the major cation and lactate is added as the Li+ salt, the formation of ApH may be limited by exchange of Ht for those cations.

ACKNOWLEDGEMENTS: We thank Dr. E. Sorensen for his helpful discussions. This work was supported by grants from the National Science Foundation (PCM 77-17652) and the National Institute of General Medical Sciences of the National Institutes of Health (GM 21648).

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REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Mitchell, P. (1973) J. Bioenergetics 4:63-91. Rosen, B.P. and Kashket, E.R. (1978)-In Bacterial Transport, B. Rosen, ed., Marcel Dekker, Inc., New York, pp. 559-620. West, I.C. and Mitchell, P. (1974) Biochem. J. 144:87-90. Schuldiner, S. and Fishkes, H. (1978) Biochemistry 17:706-711. Tokuda, H. and Kaback, H.R. (1977) Biochemistry 1x2130-2136. Lanyi, J.K. and MacDonald, R.E. (1976) 15:4608-463, Bhattacharyya, P. and Barnes, E.M. (197r J. Biol. Chem. 253:3848-3851. Niven, D.F. and MacLeod, R.A. (1978) J. Bacterial. 134:7m43. Rosen, B.P. and McClees, J.S. (1974) Proc. Natl. AcxSci. USA 71:5042-5046. Bhattacharyya, P. and Barnes, E.M. (1976) J. Biol. Chem. 251:561F5619. Golub, E.E. and Bronner, F. (1974) J. Bacterial. 119:840-843. Belliveau, J.W. and Lanyi, J.K. (1978) Arch. Biocxm. Biophys. 186:98-105. Hayashi, S., Koch, J.P. and Lin, E.C.C. (1964) J. Biol. Chem. 23mO98-3105. Tanaka, S., Lerner, S.A., and Lin, E.C.C. (1967) J. Bacterial. m42-648. Rosen, B.P. and Adler, L.A. (1975) Biochim. Biophys. Acta 38m3-36. Bradford, M.M. (1976) Anal. Biochem. z:248-254. Ordal, G.W. (1977) Nature 270:66-77. Hasan, S.M. and Tsuchiya, m977) Biochem. Biophys. Res. Commun. 78:122-128. Rhoads, D.B., Waters, F.B. and Epstein, W. (1976) J. Gen. Physiol. 67:3B-342. Padan, E., Ziberstein, D. and Rottenberg, H. (1976) Eur. J. BiocheKE:533-541.

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