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Relationship Between the Na+/H' Antiporter and Na+/Substrate Symport in Bacillus Alcalophilus (Nonalkalophilic Mutant/Efflux/Vesicles) ARTHUR A

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Relationship Between the Na+/H' Antiporter and Na+/Substrate Symport in Bacillus Alcalophilus (Nonalkalophilic Mutant/Efflux/Vesicles) ARTHUR A Proc. Nati. Acad. Sci. USA Vol. 78, No. 3, pp. 1481-1484, March 1981 Biochemistry Relationship between the Na+/H' antiporter and Na+/substrate symport in Bacillus alcalophilus (nonalkalophilic mutant/efflux/vesicles) ARTHUR A. GUFFANTI*, DUNELL E. COHN+, H. RONALD KABACKt, AND TERRY A. KRULWICH*t *Department of Biochemistry, Mount Sinai School of Medicine of the Citv of New York, New York, New York 10029; and tLaboratory of Membrane Biochemistrv, Roche Institute of Molecular Biology, Nutley, New Jersey 07110 Communicated by B. L. Horecker, November 24, 1980 ABSTRACT The Na+/H+ antiporter of the obligate alkalo- pendently isolated nonalkalophilic strains lacks both Na+/H+ phile Bacillus alcalophilus facilitates growth at alkaline pH and antiport and Na+/AIB symport activity (15); and at least a dozen precludes growth below pH 8.5. Thus, nonalkalophilic mutant revertants have regained both activities simultaneously, as well strains do not exhibit Na+/H+ antiport activity and, interestingly, such strains concomitantly lose the ability to catalyze Na+-depen- as the characteristic wild-type properties of the respiratory dent accumulation of a-aminoisobutyrate [Krulwich, T. A., Man- chain (15, 16). For these reasons, the possibility was considered del, D. G. Bornstein, R. F. & Guffanti, A. A. (1979) Biochem. that there may be a more direct relationship between the Na+- Biophys. Res. Commun. 91, 58-62]. Several other Na'-dependent translocating antiport and symport systems than generally transport systems are now documented in vesicles from the wild- thought to be the case. The experiments presented here support type strain, and it is demonstrated that these systems are defective this notion by demonstrating that mutational loss of Na+/H+ in vesicles from the nonalkalophilic mutant KM23. Surprisingly, the defect seems to result not from the loss of Na+/H' antiport antiport activity in B. alcalophilus KM23 simultaneously leads activity per se but from a pleiotropic defect in the Na+/substrate to a pleiotropic defect in Na+-dependent substrate translocation. symporters themselves. Monensin, an ionophore that catalyzes Na+/H+ exchange, does not restore respiration-driven Na+/ MATERIALS AND METHODS substrate symport in KM23 vesicles. Moreover, with KM23 ves- icles, efflux of a-aminoisobutyrate, L-malate, and L-aspartate Growth of Cells and Preparation of Membrane Vesicles. B. down their respective concentration gradients is not stimulated alcalophilus (American Type Culture Collection 27647) and the by Na+, in contrast to the observations with wild-type vesicles. nonalkalophilic derivative KM23 were grown on L-malate-con- Because monensin should ameliorate a simple defect in Na+/H+ taining media at pH 10.5 and 6.8, respectively (4, 15). Mem- antiport activity and the antiporter should notbe requiredfor Na+/ substrate symport down a concentration gradient, the results sug- brane vesicles were prepared by osmotic lysis of lysozyme-in- gest that there may be a direct relationship between the antiporter duced protoplasts (12, 17), suspended in 100 mM potassium and various Na+/substrate symporters. One possibility is that the carbonate buffer (pH 9.0) containing 10 mM MgSO4, frozen in systems share a Na+-translocating subunit. liquid nitrogen, and stored at -70°C. Transport Assays. Uptake of radioactive substrates was mea- Although many bacterial transport systems catalyze the coupled sured by filtration as described (4). The assay mixtures con- movement of protons with substrate (i.e., H+/substrate sym- tained 100 mM potassium carbonate (pH 9.0), 10 mM MgSO4, port), a substantial number catalyze Na+/substrate symport in and a given radioactive substrate at a specified concentration analogy with eukaryotic transport systems (1-6). Unlike eukar- and specific activity. Where indicated, 10 mM sodium carbon- yotes, however, most bacterial cells do not possess a primary ate was also added. Potassium ascorbate and N,N,N',N'-tetra- Na+ pump (e.g., Na+, K+-ATPase), and an overall mechanism methyl-p-phenylenediamine (TM PD) at final concentrations of proposed by Mitchell (7) that involves indirect coupling be- 20 mMl and 2 mM, respectively, were used as an artificial elec- tween various Na+/substrate symporters and an antiporter cata- tron donor system, and the reaction mixtures were gassed with lyzing H+/Na+ exchange has received strong experimental sup- water-saturated oxygen. Uptake was initiated by the addition port (2-13). By this means, Na+/substrate symport is driven of 20 ,ul of a membrane suspension containing 100 ,ug of mem- thermodynamically by the electrochemical gradient of protons, brane protein to 80 ,ul of reaction mixture. The reactions were which functions to maintain a Na+ gradient (Na+ < Na+,0) terminated by rapid dilution with potassium carbonate buffer, through the activity of a Na+/H+ antiporter. immediate filtration through nitrocellulose filters (pore diam- Because the Na+/H+ antiporter and the Na+/substrate svm- eter 0.45 pAm), and one wash with the same buffer (4). Radio- porters are presumed to be distinct, substrate-specific catalysts, activity was determined by liquid scintillation spectrometry. it is notable that nonalkalophilic mutants of Bacillus alcalophi- Carrier-mediated efflux down a concentration gradient was lus deficient in Na+/H+ antiport activity are also defective in measured as described (18-20). Vesicles were concentrated to Na+/aminoisobutyrate (AIB) symport (13-15). Although dimin- approximately 30 mg of protein per ml in 100 mM potassium ished steady-state levels of Na+-dependent AIB accumulation carbonate (pH 9.0) containing 10 mM MgSO4 and, where in- secondary to loss of antiport activity are expected, the initial dicated, 10 mM sodium carbonate. A small sample of a given rate of AIB transport is also severely altered. Furthermore, radioactive substrate was added to a specified concentration and addition of monensin, an ionophore that catalyzes Na+/H + ex- specific activity, and the suspension was incubated for 4 hr at change, does not restore AIB accumulation in the nonalkalo- 5°C to allow equilibration with the intravesicular space. Efflux philic mutant (15). Importantly, each of several dozen inde- reactions were initiated by diluting 2 ,ul of the equilibrated The publication costs of this article were defrayed in part by page charge Abbreviations: AIB, a-aminoisobutyrate; TMPD, N,N,N',N'-tetra- payment. This article must therefore be hereby marked "advertise- methyl-p-phenylenediamine. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 1481 Downloaded by guest on September 25, 2021 1482 Biochemistry: Guffanti et alPProc. Natl. A.ad. Sci. USA 78 (1981) vesicle suspension into 2 ml of a given buffer, followed by im- pendent AIB accumulation is apparent from the onset of the mediate dispersion with a Vortex mixer. After incubation for transport reaction (i.e., as early as 5 sec). As found previously various times at 250C, the vesicle suspensions were filtered with intact cells (15), KM23 vesicles exhibit appreciable AIB through 0.45 gm nitrocellulose filters and the retained vesicles uptake at pH 7.0, but uptake at this pH is not Na+ dependent were washed with 2 ml of the reaction buffer. Radioactivity (data not shown). Thus, KM23 has an AIB porter that is func- retained on the filters was determined by liquid scintillation tional at neutral pH, but it is no longer Na+ dependent. It is spectrometry. The percentage of solute retained was calculated also evident from the data in Fig. 1 that addition of monensin by comparison to zero-time points obtained as described to KM23 vesicles has no effect whatsoever on AIB accumula- (18-20). In some experiments, there was an initial, rapid loss tion. This observation is important because it is expected from of solute that is unrelated to carrier-mediated efflux (18-20). straightforward chemosmotic considerations that the ionophore The half-time of efflux (tij2) was calculated from the first-order would provide the system with the ability to exchange internal rate of efflux after an initial rapid loss of solute. Na+ for external H+ and thus restore Na+-dependent AIB ac- Protein Assay. Protein was determined by the method of cumulation to some extent at least in these vesicles. The lack Lowry et al. (21), using egg white lysozyme as standard. of a restorative effect of monensin in this regard is not due to Chemicals. L-[U-'4C]Malic acid (55 mCi/mmol) was ob- an inability of the ionophore to function at high pH (4). Also, tained from Amersham/Searle, and L-[2,3-3H]aspartic acid (25 previous work (4) indicates that monensin catalyzes Na+/H+ Ci/mmol) and a-amino[1-'4C]isobutyric acid (53.2 mCi/mmol) exchange even in the presence of high K+ concentrations (i.e., were from New England Nuclear (1 Ci = 3.7 x 1010 becque- under the conditions described in Fig. 1). As an added control, rels). Monensin was generously provided by R. L. Hamill (Eli however, KM23 vesicles were prepared in 100 mM sodium car- Lilly). All other materials were reagent grade of highest avail- bonate buffer at pH 9.0, and monensin was added at concen- able purity. trations ranging from 0.1 to 2.0 ,ug/ml. No AIB uptake was ob- served in the presence of ascorbate/TMPD at any of the RESULTS monensin concentrations employed. Because the protonmotive force is relatively low in alkalo- As shown in Fig. 1, in the presence of ascorbate/TMPD and philic bacteria (4, 14), it is reasonable to expect that such bac- Na', membrane vesicles from wild-type B. alcalophilus take up teria utilize the electrochemical Na+ gradient for active trans- AIB rapidly and achieve a steady-state level of accumulation in port, and several Na+-dependent symport systems have been approximately 10 min (not shown), whereas in the absence of described in alkalophiles (4, 14, 22, 23).
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