Requirement for Membrane Potential in Active Transport of

Transcription

1 JOURNAL OF BACTERIOLOGY, Jan. 1979, p /79/ /05$02.00/0 Vol. 137, No. 1 Requirement for Membrane Potential in Active Transport of Glutamine by Escherichia coli CHARLES A. PLATEt Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts Received for publication 24 July 1978 The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mm exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells. Certain active transport systems of Escherichia coli, such as those for glutamine and findings led to the suggestion that glutamine these compounds is apparent (2, 3, 27). These galactose, require substrate-specific periplasmic transport is driven by phosphate bond energy binding proteins for activity and are sensitive to derived from ATP, a suggestion later generalized osmotic shock (10, 14). Other transport systems, to include all binding protein-dependent transport systems in E. coli (3). including those for proline and lactose, do not have a binding protein component and are resistant to osmotic shock. Considerable evidence sion may have to be modified. We have previ- More recent findings indicate that this conclu- indicates that these latter transport systems are ously shown that both the proton-motive forcecoupled transport of proline and the ATP-de- coupled to an electrochemical gradient of protons (the proton-motive force) which is comprised of a ph gradient (interior alkaline) and a nearly the same extent by colicin K in an ATPpendent transport of glutamine are inhibited to transmembrane electrical potential (interior ase-deficient mutant of E. coli (17). The intracellular ATP levels in this mutant were shown negative) (7, 9, 19, 26). The proton-motive force arises either as a consequence of electron transport or through the hydrolysis of ATP mediated ment, indicating that something in addition to to increase as a consequence of colicin K treat- by the energy-transducing adenosine triphosphatase (ATPase) (9). tamine transport system. The data to be pre- ATP is required for the functioning of the glu- The proton-motive force per se does not appear to be sufficient to drive the binding protein- in addition to ATP a normal membrane potensented in this communication demonstrate that dependent transport systems, an observation tial is required for the functioning of the glutamine transport system. first made by Berger (2) for glutamine transport. Glutamine transport, unlike proline transport, is Weiss and Luria (30) have found that an early sensitive to inhibition by arsenate and cannot be consequence of treating E. coli cells with colicin driven by respiratory substrates, such as D-lactate or ascorbate-phenazine methosulfate, in with little or no effect on the ph gradient. These K is the abolition of the membrane potential mutants of E. coli lacking a functional energytransducing ATPase. Glutamine transport also hibited by colicin K even under conditions where findings explain why glutamine transport is in- appears to be less sensitive than proline transport to such proton conductors as carbonyl cy- MATERIALS AND METHODS intracellular ATP levels remain high (17). anide-p-trifluoromethoxyphenylhydrazone and Bacterial srain and media. E. coli CJ30 2,4-dinitrophenol, although some inhibition by [lacz(am) thp(am) strr] was the parental strain used t Present address: Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL tive of CJ30 that was isolated after penicillin selection. in this study. Strain CJ36 is a spontaneous ilv deriva Strains CJ37 [lacz(am) trp(am) strr unca401] and 221

2 222 PLATE CJ38 [lacz(am) trp(am) strr uncb401] were isolated after P1 transduction with strain CJ36 as recipient and E. coli strains AN120 (arge3 thi-1 strr unca401) (4) and AN480 (frd-1 enta uncb401) (5) as donors, respectively. Cultures were routinely grown at 370C in Ozeki medium base (16) supplemented with glucose (0.4%), tryptophan (50 ug/ml), and thiamine (1.0 Lg/ml). Tris-EDTA treatment of cells. To make cells permeable to triphenylmethylphosphonium ion (TPMP+) and valinomycin, they were treated with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA) as described by Szmelcman and Adler (29). After Tris-EDTA treatment the cells were washed twice with cold 0.1 M sodium phosphate (ph 7.0) and were then suspended in 0.01 M sodium phosphate (ph 7.0) to an optical density at 500 nm of approximately 8. These cells were kept on ice until used. TPMP+ and glutamine uptake. To measure TPMP+ uptake, 0.3 ml of cell suspension was added to a flask containing 0.3 ml of 0.01 M sodium phosphate (ph 7.0) supplemented with glucose (22 mm), MgSO4 (20 mm), chloramphenicol (100,ug/ml), and, where indicated, potassium ion (40 mm) and valinomycin (10,tM). Potassium ion was added as 1.0 M potassium phosphate (ph 7). The suspension was incubated with shaking at 270C for 4.5 min. Na+ tetraphenylboron (2,uM final concentration) was then added, followed after 30 s by [3H]TPMP+Br- (58.5,uCi/,umol) to a final concentration of 8.7,uM. At the indicated times 0.1-ml samples were removed, and the cells were collected on Celotate filters (Millipore type EH, 0.5-,um pore size) and washed with 4 ml of 0.1 M LiCl. After drying, the filters were placed in Omnifluor-toluene and radioactivity was measured in a Beckman liquid scintillation spectrometer. Glutamine uptake was measured in a similar fashion except that ['4C]glutamine (10.6,Ci/pmol) was added to a final concentration of 8.5,uM, and the cells were collected on nitrocellulose filters (Matheson-Higgins, Inc.; 0.45-,um pore size). Nonspecific binding of ['4C]glutamine and [3H]- TPMP+Br- to the filters was measured in each experiment, and these blank values were subtracted from the uptake data obtained. ATP deterninations. A sample (1.25 ml) of Tris- EDTA-treated cells in 0.01 M sodium phosphate (ph 7) containing 20 mm potassium was added to a flask containing 0.01 M sodium phosphate (ph 7) supplemented with potassium (20 mm), glucose (22 mm), MgSO4 (20 mm), and chloramphenicol (100 ulg/ml). After this suspension was incubated at 27 C with shaking for 4.5 min, a 0.5-ml sample was removed to a tube on ice containing 0.13 ml of 2 N perchloric acid. Thirty seconds after removal of this sample, either 10 pd of 1 mm valinomycin in 100% ethanol (final valinomycin concentration, 5 gm) or 10 pl of 100% ethanol was added to the cell suspension; then, at the indicated times, 0.5-ml samples were removed to tubes on ice containing perchloric acid. After a minimum of 10 min on ice, the samples were centrifuged to remove acidinsoluble material. The supernatants were decanted to clean tubes and were neutralized with 2 N KOH. After centrifugation to remove the insoluble potassium perchlorate, the supernatants were analyzed for ATP content using firefly lantern extract as described by Feingold (6). A standard curve for ATP was constructed for each experiment from supernatants prepared from samples without cells and containing a known amount of exogenously added ATP. Protein determination. Protein was determined by the method of Lowry et al. (15), using bovine serum albumin as a standard. Chemicals. L-[U-'4C]glutamine was obtained from New England Nuclear Corp., and [3H]TPMP+Br- was a gift from H. R. Kaback. Valinomycin and firefly lantern extract were purchased from Sigma Chemical Co. Na' tetraphenylboron was a gift from C. Walsh, Massachusetts Institute of Technology. All other chemicals used were reagent grade. RESULTS Effect of valinomycin on glutamine uptake. The objective of this study was to determine the effect of reducing the membrane potential on the ATP-dependent transport of glutamine. Addition of valinomycin to Tris- EDTA-treated cells of E. coli in the presence of sufficiently high levels of exogenous potassium lowers the membrane potential without reducing the ph gradient established by the cells (18). The effects of valinomycin and potassium on glutamine transport are shown in Fig. 1. Addition of valinomycin to Tris-EDTA-treated E. coli cells in the presence of 20 mm exogenous potasium resulted in the complete inhibition of glutamine uptake. This inhibition was not due to valinomycin-potassium complex directly interacting with the glutamine transport system, since neither valinomycin in the absence of potassium (Fig. 1) nor valinomycin in the presence of low levels of potassium (50,uM, data not shown) caused this inhibition. The extent to which the membrane potential was reduced under these conditions was estimated by measuring the uptake of radiolabeled _ E0 41 S 1..0_ z 0. Z.5 - K+ 20mM K+ +Valinomnycin / * - Valinomycin J. BACTERIOL. >t -Valinomycin + Val inomyc in Ȯ I_ * * MINUTES FIG. 1. Effect of valirnmycin on glutamine transport in the absence andpresence of exogenous potassium ion. Experimental details are described in the text. 0

3 VOL. 137, 1979 TPMP+, a lipophilic cation. TPMP+ distributes itself passively between the cells and the medium in accordance with the membrane potential (1, 8, 22). In the absence of exogenous potassium valinomycin had little effect on the level of TPMP+ accumulated by the cells, whereas in the presence of 20 mm potassium TPMP+ uptake was markedly diminished (Fig. 2). Effect of valinomycin and potassium on intracellular ATP levels and glutamine uptake in normal and ATPase-deficient strains. Treating cells of E. coli with valinomycin in the presence of 20 mm exogenous potassium also resulted in a marked reduction in the intracellular levels of ATP (Fig. 3A). Control experiments showed that valinomycin in the absence of exogenous potassium had no effect on intracellular ATP levels (data not shown). Because of the ATP requirement for glutamine uptake by E. coli cells (2) it was necessary to determine the effects of valinomycin and potassium on glutamine uptake under conditions in which the intracellular ATP levels remained high. This was done by using mutants of E. coli defective in the Ca2+, Mg2+-activated ATPase complex. The E. coli unca derivative used is defective in the Fl portion of the ATPase complex and has greatly diminished levels of ATPase activity (4). The E. coli uncb derivative has been shown to possess a normal Fl portion of the ATPase complex, but it is deficient in the membrane-associated FO portion and is unable to carry out oxidative phosphorylation or to transfer the energy derived from ATP hydrolysis to energy-consuming reactions such as the energy-dependent transhydrogenase (5). Addition of valinomycin to the unca and uncb mutants in the presence of 20 mm potassium had little effect on the intracellular ATP levels in either strain (Fig. 3B and C). With the ATPase-deficient MEMBRANE POTENTIAL AND GLUTAMINE TRANSPORT 223 mutants, there- MINUTES FIG. 2. Effect of valinomycin on TPMP' uptake in the absence and presence of exogenous potassium ion. Experimental details are described in the text. 10A 0 A 5 Control C -6 ~~~~~~~~~~~~~~~Control ocontrol UT InOycin D V oc -4 IG 3. Efeto alinomycin o h nrclua ATP levels of a normal strain of E. coli and two unc derivatives of this strain. Experimental details are described in the text. fore, it was possible to reduce the membrane potential without reducing the intracellular levels of ATP. Figure 4 shows that valinomycin in the presence of 20 mm potassium affects the membrane potential of the ATPase-deficient strains (as measured by TPMP+ uptake) in analogous fashion to the normal strain. In the presence of exogenous potassium (20 mm), valinomycin also inhibited glutamine accumulation by both unc mutants approximately 80%, relative to control cultures receiving no valinomycin (Fig. 5). DISCUSSION Whereas it seems clear that ATP, or some metabolite derived from ATP, plays a role in the transport of glutamine by E. coli cells (2, 3), the results of the present study indicate that membrane potential is also important to this transport system. Findings from other laboratories are compatible with this conclusion. Lieberman and Hong (12) concluded from studies on an ecf(ts) mutant of E. coli that the osmotic shock-sensitive transport systems of E. coli have a requirement in addition to ATP and suggested that this requirement is a functional ecf gene product. At restrictive temperatures this mutant exhibits deficiencies in both protonmotive force-linked and ATP-dependent transport systems, and the shift from permissive to restrictive temperatures results in a twofold increase in intracellular ATP levels. It has been reported, however, that at restrictive temperatures a strain of E. coli carrying this mutation is incapable of maintaining a normal membrane potential (13). While my results do not exclude the possibility of a direct role for the ecf gene product in the coupling of energy to the ATPdependent transport systems, they suggest the alternative possibility that the deficiency in the ATP-dependent transport systems manifested in the ecf(ts) mutant at high temperature is a consequence of the loss of the membrane potential under these conditions.

4 224 PLATE o MINUTE S FIG. 4. Effect of valinomycin on TPMP+ uptake by an unca and an uncb mutant of E. coli. Potassium ion was present in the incubation mixture (see the text) at a concentration of20 mm..ti PO. I.0 u,nc A unc B 1.8 /- Volinomycin! 0 go).4 -J/ oio-yi o9 / + Volimmfycin MINUTE 0N S A ' - o v I!! I+VaIinl"Ionye FIG. 5. Effect of valinomycin on glutamine uptake by an unca and an uncb mutant ofe. coli. Potassium ion was present in the incubation mixture (see the text) at a concentration of20 mm. That glutamine transport requires a membrane potential is also consistent with findings presented by Singh and Bragg (27, 28). These investigators, using cytochrome-deficient mutants of E. coli (27) and Salmonella typhimurium (28), have shown that in the absence of electron transport glutamine transport is dependent upon a functional energy-transducing ATPase. In the absence of electron transport, the membrane potential and the ph gradient are maintained by the hydrolytic action of the ATPase coupled with the pumping of protons out of the cell. Inactivation of the ATPase under these conditions would thus result in the abolition of the membrane potential with the concomitant inhibition of glutamine transport. No attempt has been made in this study to establish a quantitative relationship between the magnitude of the membrane potential and the levels of glutamine uptake obtained. Although the uptake of TPMP+ serves as a good indicator of changes in the membrane potential, arriving J. BACTERIOL. at quantitative estimates of membrane potential from the TPMP+ distribution ratio requires accurate determinations of the intracellular space and the assumption that the internal TPMP+ is free in solution. Qualitatively, however, one can interpret the data in Fig. 2 and 4 as indicating that valinomycin in the presence of exogenous potassium lowers the membrane potential with the concomitant inhibition of glutamine uptake. A point that deserves emphasis is that both membrane potential and ATP, or a compound derived from ATP, are required for the normal uptake of glutamine by E. coli cells. Neither component alone is sufficient to energize this trasport system. The results of the present study and data obtained using colicin K (17) have shown that reducing or abolishing the membrane potential under conditions where intracellular ATP levels are unaffected or increased results in the inhibition of glutamine uptake. Conversely, Flagg and Wilson (7) have shown that imposing a membrane potential by the inward diffusion of thiocyanate ion into energy-depleted E. coli cells under conditions where ATP synthesis is blocked is not sufficient to drive glutamine uptake, whereas concentrative uptake of proline does occur. Thus any model proposed for the glutamine transport system must take both of these elements into account. The membrane potential may function in glutamine transport in a manner similar to that which has been postulated for the lactose transport system of E. coli. Studies in the laboratories of Kaback (20, 21, 23, 25) and Hinkle (11) with the fluorescent lactose analogs dansylgalactosides, which bind to the lactose carrier but are not transported, indicate that imposition of a membrane potential increases the number of dansylgalactoside binding sites on the exterior surface of the membrane. It has been postulated that this could be accounted for if the lactose carrier were negatively charged-the membrane potential would drive this charge to the positive side of the membrane and expose the substrate binding site (11). In the case of the glutamine carrier, an additional feature of such a model might be either the binding of ATP and its functioning in a regulatory capacity (12), or phosphorylation of the carner (2), possibly to provide the net negative charge. The possibility that other osmotic shock-sensitive, ATP-dependent transport systems of E. coli also require membrane potential for normal functioning is currently under investigation. Preliminary studies on the ATP-dependent galactose and f3-methyl-galactoside transport system (31) indicate that reducing the membrane potential either by treatment with colicin K (30) or

5 VOL. 137, 1979 MEMBRANE POTENTIAL AND GLUTAMINE TRANSPORT 225 with valinomycin and potassium results in a marked inhibition of both of these transport systems (C. Plate, unpublished data). Thus the requirement for membrane potential does not appear to be restricted to the glutamine transport system and may be a factor in others, if not all, of the shock-sensitive, ATP-dependent transport systems of E. coli. ACKNOWLEDGMENTTS I thank S. E. Luria, in whose laboratory this work was carried out, for many helpful discussions. Suggestions by V. L. Clark, C. Kayalar, J. L. Suit, and M. Weiss are also appreciated. This work was supported by grants to S. E. Luria from the National Science Foundation (no. PCM ) and the National Institute of Allergy and Infectious Diseases (Public Health Service grant 5-RO1-AI03038). LITERATURE CITED 1. Bakeeva, L. E., L. L. Grinius, A. A. Jasaitis, V. V. Kuliene, D. 0. Levitsky, E. A. Liberman, I. I. Severina, and V. P. 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