Proc. Nati. Acad. Sci. USA Vol. 75, No. 7, pp. 3216-3219, July 1978
Biochemistry
Identification of the structural proteins of an ATP-driven potassium transport system in Escherichia coli (inner membrane proteins/Kdp transport system/kdp transducing phage/amber mutations/periplasmic protein)
LAIMONIs A. LAIMINS*, DAVID B. RHOADS*, KARLHEINZ ALTENDORFt*, AND WOLFGANG EPSTEIN*§ * Department of Biophysics and Theoretical Biology, § Department of Biochemistry, University of Chicago, Chicago, Illinois 60637; and t Institut fur Biologie, Universitit Tubingen, D 7400 Tfibingen 1, Germany
Communicated by Albert Dorfman, May 5, 1978
ABSTRACr The three structural proteins of the ATP-driven Kdp potassium transport system of Escherichia coli [Rhoads, D. B., Waters, F. B. & Epstein, W. (1976) J. Gen. PhysioL 67, 325-341] have been identified and found to be located in the inner membrane. The high-affinity repressible Kdp system is one of four potassium transport systems in E. coli. The Kdp proteins were identified both in growing cells as well as in heavily UV-irradiated cells infected with transducing phages carrying the kdp operon. Although all previously identified ATP-driven transport systems of Gram-negative bacteria have been shown to contain a periplasmic protein component, no evidence was found for such a component or for an outer membrane component of the Kdp system. The molecular weights of the three inner membrane proteins, KdpA, KdpB, and KdpC, were determined to be 47,000, 90,000, and 22,000, respectively. ATP or another high-energy phosphate compound serves as the energy source for a number of active transport systems in bacteria (1, 2). The characterization of ATP-driven systems in bacteria remains incomplete. Many of these transport systems in the enteric bacteria have a soluble, periplasmic "binding" protein that binds the substrate with high specificity and affinity (3-6). Such binding is believed to be the first step in transport (7) and is followed by translocation across the inner bacterial membrane by one or more proteins specific to the transport system. Although these additional proteins, presumably membrane proteins, have been genetically defined for several systems (8-10), they have not been biochemically identified for any bacterial system. Escherichia coli has a high-affinity repressible transport system for potassium, designated the Kdp system (11), which is ATP-driven (12). The function of this system depends on the expression of the four clustered kdp genes (13) comprising the kdpD regulatory gene and the kdpABC operon (14). Here we report the identification of the three proteins coded by the kdpABC operon. All are inner membrane proteins. The Kdp system does not appear to have a periplasmic protein component.
is dependent on the Kdp system up to an external K+ concentration of approximately 20 mM (11). The Kdp transducing phage, Xpkdp-1, has been reported to carry all three structural genes of the kdpABC operon (14). All transducing phages carry the XcI857 mutation. Phages carrying kdp amber mutations were prepared by lysogenizing a host carrying the desired mutation with Xpkdp-1, inducing the prophage by shifting the culture to growth at 420, and screening for Kdp- phages by the ML plate method (14). The genotype of the mutant phages was confirmed by their ability to complement a nonsupressible kdp point mutation in the same gene in a suppressing host but not in a nonsuppressing host. Growth Media and Conditions. Cells were grown with shaking at 370 in medium containing glucose (4 g/liter) as carbon and energy source. The phosphate-buffered media, referred to by K+ concentration in mM, have been described (15). Growth in K15 fully represses the Kdp system except in constitutive strains TL1000 and TL1007 which express the Kdp system at 12% of the fully derepressed rate due to the presence of the kdpDl7 mutation. All TK and TL strains express the Kdp proteins at a high rate when shifted from K15 medium to K5 or K10 medium, whereas wild-type strain Ymel expresses the Kdp system at a high rate only when grown in media containing less than 100 IAM K+. Labeling of Phage-Coded Proteins. Labeling was per-
formed by the method of Ptashne (16), with the modification of McEntee et al. (17). The cells were grown and irradiated in KLlS medium. After a 60-min recovery in K15 medium, the cells were infected and labeled for 60 min at 370 in either K10 or K115 medium, as indicated, with 10 ;tCi of "4C-labeled amino acid mixture (Schwarz/Mann) per 109 cells. Labeling Growing Cells. After derepression of the Kdp system by a shift from K1 15 medium to K5 medium, the cells at a concentration of 5 X 108/ml were labeled with 14C(labeled (0.3 ,Ci/ml) amino acid mixture (Schwarz/Mann). Labeling occurred over the interval from 15 to 135 min after transfer to K5.
Analysis of Labeled Proteins. Sodium dodecyl sulfate/ polyacrylamide electrophoresis and photofluorogram analysis were performed as described (17).
MATERIALS AND METHODS Bacteriophages and Strains. The strains used are listed in Table 1. In addition to the Kdp system, three other K+-transport systems are genetically defined in E. coli. In all TK and TL strains shown in Table 1, the function of the other two saturable K+-transport systems, TrkA and TrkD (11), is abolished by mutations (15). This facilitates genetic and transport studies because, in trkA trkD mutants, both growth and K+ transport
RESULTS Identification of Xpkdp-1 Coded Proteins. Genes introduced by a transducing bacteriophage are preferentially expressed when a host defective in DNA repair is irradiated with a high dose of UV, followed by phage infection. If, in addition, the host is lysogenic for Xind-, a noninducible mutant of X,
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
* Present address: Lehrstuhl fur Biochemie der Pflanzen, Ruhr-Universitat Bochum, D 4630 Bochum 1, Germany.
3216
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Biochemistry:
Laimins et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Bacterial strains* and bacteriophages Designation TL1000 TL1007 TL1011 TK2104/4 TK2121/21 TK2130/30 TK2131/31 Ymel xpkdp-l Xpkdp-l-A21 Xpkdp-1-B30 Xpkdp-l-C31
B
Pertinent genotypet
kdpDl 7 uvrA (Xind -) kdpDl 7 uvrA supD (Xind) kdpDll uvrA (Xind-) kdpA4 nadA with F100-kdpA4 kdpA21 nadA with F100-kdpA21 kdpB30 nadA with F100-kdpB30 kdpC31 nadA with F100-kdpC31
A
_
kdp+
c
Xc1857 kdpABC+ Xc 1857 kdpA21 Xc1857 kdpB30 Xc1857 kdpC31
* The TL and TK strains are also trkA401
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g c f e d b FIG. 2. Identification of the genes coding for the Kdp proteins. Labeling of proteins and electrophoresis were performed as for lane c of Fig. 1; a photofluorogram of the gel is shown. Nonsuppressing strain TL1000 was infected with Xpkdp-1 carrying the indicated amber kdp mutations: lane a, kdpA21; lanes b and e, kdpB30; lanes c and g, kdpC31. To show restoration of the missing band by an amber suppressor, supD strain TL1007 was infected with Xpkdp-1 carrying: lane d, kdpB30; lane f, kdpC31. a
trkDl.
t The listed kdp alleles have been characterized as follows: kdpA4,
missense; kdpA21, amber; kdpB30, amber; kdpC31, amber; kdpDll, missense; kdpD17, missense resulting in partial constitutivity of the Kdp system.
expression of all X genes under control of X repressor is blocked. Under these conditions, bacterial genes carried by the transducing phage and not under repressor control, as well as the X repressor gene itself, are preferentially expressed. By using this protocol, the bacterial proteins coded by Xpkdp-l were iden-
tified (Fig. 1). When protein labeling was performed in K10 medium to derepress the kdp genes on the phage, four prominently labeled proteins were seen (Fig. 1, lane c). Three of these proteins, marked A, B, and C, were much less prominent when labeling was performed in K115 medium (Fig. 1, lane b) which, in this partially constitutive host, reduces expression of the kdp genes to 12% of the maximum rate. The three marked proteins were not seen in the uninfected control (Fig. 1, lane a), nor were they found after infection with wild-type X. These results suggest that the proteins marked A, B, and C are the products of the three genes of the kdpABC operon. Infection of host
I,
3217
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A
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c a b FIG. 1. Identification of the Kdp proteins. Proteins synthesized after infection of UV-irradiated strain TL1000 with Xpkdp-1 were labeled with radioactive amino acids and separated electrophoretically on a gel containing 20% acrylamide and 0.07% methylene bisacrylamide. A photofluorogram of the gel is shown; migration is from top to bottom. Each sample contained 50,000 cpm. (a) Uninfected cells, labeled in K115 medium; (b) Xpkdp-l-infected cells, labeled in K115 medium; (c) Xpkdp-l-infected cells, labeled in K10 medium. Letters at the right identify the Kdp proteins and the X repressor.
strain TL1011, whose kdpDll mutation prevents expression of the kdp operon (13, 14), did not result in synthesis of proteins A, B, and C, demonstrating that synthesis of these proteins is under kdpD control. The fourth protein was identified as the X repressor. It was made after infection with wild-type X (data not shown) and its molecular weight of 27,000, estimated by using T4 proteins as standards (18), is consistent with that of the X repressor (19). The genes coding for each of the proteins were identified by examination of the proteins made after infection with mutant Xpkdp-1 phages carrying kdp amber mutations. In each infection, one of the three proteins was not synthesized (Fig. 2, lanes a-c). To make sure that absence of a protein was not due to some artifact, we examined proteins synthesized upon infection of hosts carrying an amber suppressor mutation with the kdp amber phages. The reappearance of the proteins in these hosts (Fig. 2, lanes d and f) confirmed that we were observing an effect due to chain termination by the amber mutations. The data show that kdpB codes for the largest protein (designated B), kdpA codes for the protein of intermediate size (marked A), and protein C (with the lowest monomer molecular weight) is coded for by kdpC. Identification in Growing Cells. The same three Kdp proteins were synthesized at a high rate in cells derepressed for the Kdp system. Membrane proteins of kdp diploid strains labeled under Kdp derepressing conditions are shown in Fig. 3. All three Kdp proteins were synthesized in the kdpA4 missense mutant strain, shown in lane g. The protein product of a particular gene is absent in the strain carrying an amber mutation in that gene (lanes d-f). We attribute the lack of a visible KdpC protein and the much decreased amounts of the KdpB protein in the strain with the kdpA21 amber mutation (Fig. 3, lane f) to a polar effect. The kdp operon is transcribed from kdpA to kdpC (14), so that a chain-terminating mutation near the promoter end of the kdpA gene will be polar on expression of kdpB and kdpC. The relatively small amounts of the KdpB and KdpC proteins made after infection with the kdpA21 phage (Fig. 2, lane a) suggest that this mutation is also polar when the kdp operon is expressed in a heavily UV-irradiated host. All three Kdp proteins were stable; there was no apparent loss of label from any of the three proteins during a 2-hr chase in
3218
Biochemistry: Laimins et al. awk4w
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 3. Identification of the Kdp proteins among membrane proteins of growing cells. Strains examined were homozygous kdp diploids carrying kdp point mutations. The separating gel contained 15% acrylamide and 0.4% methylene bisacrylamide. Membrane proteins of the kdpA4 mutant strain: lane a, cells labeled under Kdp repressing conditions; lane b, cells labeled under Kdp derepressing conditions and subjected to a 2-hr chase in nonradioactive medium during growth under Kdp repressing conditions; lane c, outer membrane fraction of cells labeled under Kdp derepressing conditions and separated as described by Ito et al. (20). Membrane proteins labeled under Kdp derepressing conditions in strain with indicated mutation: lane d, kdpC31; lane e, kdpB30; lane f, kdpA21; lane g, kdpA4.
which the cells were grown under repessing conditions after labeling (Fig. 3, lane b). A control showing that none of the Kdp proteins was synthesized under repressing conditions is also included (lane a). The molecular weights of the Kdp proteins were estimated by comparing their mobilities in sodium dodecyl sulfate/ polyacrylamide gels with those of protein standards in two similar experiments (Fig. 4). Comparative plots of the logarithm of molecular weight as a function of migration distance gives values of 85,000 and 95,000 for KdpB, 47,000 and 48,000 for KdpA, and 22,000 and 23,000 for KdpC. Values obtained in a similar experiment for Xpkdp-1 coded Kdp proteins using T4 early proteins (18) as standards led to estimates of 96,000, 46,000, and 21,000. Membrane Association. All three proteins of the kdpABC operon were firmly associated with the inner membrane of E. coli. Examination of separated inner and outer membranes from a wild-type strain grown either under Kdp repressing or derepressing conditions showed that all three proteins were in the inner membrane (Fig. 5). There was no trace of any of these proteins in the outer membrane fractions shown in this experiment or in the outer membrane fraction of mutant kdpA4 (Fig. 3, lane c). We found no evidence for a periplasmic protein component of the Kdp system, despite considerable effort to identify such a component. Neither one- nor two-dimensional gel electrophoretic analysis (23) of cold osmotic shock-releasable proteins (24) revealed any protein whose behavior was consistent with that of a protein coded for by or regulated in parallel with the
2
6 4 Relative migration distance
8
10
FIG. 4. Determination of the monomer molecular weight of the Kdp proteins. Shown are the results of two experiments in which migration of the Kdp proteins and of protein standards were compared. Protein standards and radioactively labeled Kdp proteins made in a diploid kdpA4 mutant were separated in the presence of sodium dodecyl sulfate as in the experiment of Fig. 3. The standards were detected by staining with Coomassie blue and the Kdp proteins, by fluorography. Protein standards and their molecular weights (X10-3) were: phosphorylase B, 92; bovine serum albumin, 67; glutamate dehydrogenase, 53; creatine phosphokinase, 40.5; carbonic anhydrase, 30; soybean trypsin inhibitor, 21.5; f3-lactoglobulin, 18.3; lysozyme, 14.4. The estimates are 95,000, 47,000, and 23,000 from the curve at the left and 85,000, 48,000, and 22,000 from that on the right for the B, A, and C proteins, respectively.
kdp genes. The stability of the Kdp inner membrane proteins in a chase experiment (Fig. 3, lane b) rules out the possibility that any serve as precursors of soluble proteins. Similar chase experiments with Xpkdp-l-coded proteins showed no change in membrane association or size during a 12-hr chase in nonradioactive medium. DISCUSSION All three structural proteins of the Kdp transport system are here shown to be stable inner membrane proteins. No periplasmic protein or outer membrane protein component was found for the Kdp system. The approximate monomer molecular weights of these proteins are 47,000, 90,000, and 22,000 for the KdpA, KdpB, and KdpC proteins, respectively. Because
A
1x
0
FIG. 5. The Kdp proteins are located in the inner membrane. Strain Ymel was grown in K5 medium to repress or in KO.05 medium to derepress the Kdp system. Isolated inner and outer membrane fractions from each of these cultures were obtained by the method of Osborn et al. (21), subjected to electrophoresis (22), and stained with Coomassie blue. Two amounts of each fraction containing approximately 50 and 80 Ag of protein were analyzed: lanes a and c, inner membranes, Kdp derepressed; lanes b and d, inner membrane, Kdp repressed; lanes e and g, outer membrane, Kdp derepressed; lanes f and h, outer membrane, Kdp repressed.
Biochemistry:
Proc. Natl. Acad. Sci. USA 75 (1978)
Laimins et al.
other ATP-driven transport systems in enteric bacteria have a periplasmic protein (3-6), we made an extensive search for such a component of the Kdp system. Evidence reported earlier suggesting a periplasmic protein component included (i) a moderate loss (30%) of Kdp transport activity after osmotic shock, and (ii) a protein released by cold osmotic shock apparently dependent on the kdp genotype of the strain (12). The studies presented above show the previous suggestion to be incorrect. Gel electrophoretic analysis of proteins released by cold osmotic shock from mutant and wild-type cells grown under different conditions demonstrate that no periplasmic protein is coded by the kdpABC operon or is regulated in parallel with the Kdp system. The reduction in Kdp transport activity after osmotic shock is most likely a nonspecific effect as noted by others in systems that do not have periplasmic proteins (25). Periplasmic proteins function as the initial substrate-binding component of other ATP-driven transport systems. Because the Kdp system does not appear to have such a protein, initial substrate binding must occur on the inner membrane. We have sought genetic evidence on the binding site by isolating mutants with an altered Km for K+. Preliminary studies of such mutants suggest that the 47,000 molecular weight KdpA protein represents the substrate binding site. The Kdp system resembles the Na+, K+-ATPase that transports these two cations across the plasma membrane of animal cells. The two subunits of this ATPase have monomer molecular weights of approximately 95,000 and 60,000 (26, 27), similar to the sizes of the KdpB and KdpA proteins, respectively. There are some striking differences as well: (i) The Kdp system has a third small subunit, the KdpC protein. (ii) The Kdp system does not appear to perform K+ uptake coupled to Na+ extrusion characteristic of the animal cell system (28). The rate of K+ uptake by bacteria suspended in buffer containing less than 20 ,uM Na+ is similar to that of cells in 100 mM Na+ buffer (12). Bacteria use a separate system to pump Na+ out, the proton Na+ antiporter (29), rather than a single system, as animal cells do, for both transport functions. The Kdp system may couple K+ uptake to extrusion of another cation such as a proton; alternatively, it may be an electrogenic process that depends on other ionic movements to maintain charge balance. (iii) The Km of the Kdp system is 2 ,gM, almost 3 orders of magnitude lower than the Km of the Na+,K+-ATPase for K+ (30). Accompanying this low Km is the ability to maintain the very high transmembrane concentration ratio for K+ of 4,000,000:1 (11). (iv) The Kdp system is a repressible transport system, made only when the other transport systems are unable to meet the cell's K+ needs. In contrast, the Na+,K+-ATPase is always needed to maintain the required cation content of animal cells. The identification of the Kdp proteins makes this system amenable to biochemical investigation. Because all identified components of the Kdp system are membrane bound, we expect this system to resemble the transport ATPases of animal cells in requiring only membrane-bound components to perform substrate translocation and energy coupling. Kobayashi and coworkers (31) have recently described an ATP-driven Ca2+ transport system from Streptococcus fecalts. Because this system is active in vesicles, it must require only membrane-bound components. The only well-characterized bacterial transport system in which high-energy phosphate bond energy is used, the phosphoenolpyruvate-dependent phosphotransferase system, requires two soluble proteins in addition to membrane
3219
components to perform transport energized by phosphoenolpyruvate (32). We anticipate that the ability to perform a full range of genetic manipulations, in addition to applying biochemical techniques, will be a significant advantage in elucidating the molecular mechanism of K+ transport by this system. The authors thank B. Ewersmeyer-Wenk, G. Heinze.lmann, and J. E. Hesse for excellent technical assistance, K. McEntee for advice in experiments with Xpkdp-l, and W. Boos for advice on two-dimensional gel electrophoresis. This work was supported by financial assistance from Prof. Dr. V. Braun, by traineeship awards to L.A.L. and D.B.R. from National Institutes of Health Training Grant GM780, and by the following research grants: PCM75-14016 from the National Science Foundation, GM22323 from the National Institutes of Health, and SFB 76 from the Deutsche Forschungsgemeinshaft. 1. Berger, A. E. (1973) Proc. Nati. Acad. Sci. USA 70, 15141518. 2. Simoni, R. D. & Postma, P. W. (1975) Annu. Rev. Biochem. 44, 523-551. 3. Cowell, J. L. (1974) J. Bacteriol. 120, 139-145. 4. Curtis, S. J. (1974) J. Bacteriol. 120, 295-303. 5. Berger, E. A. & Heppel, L. A. (1974) J. Biol. Chem. 249, 7747-7755. 6. Boos, W. (1974) Curr. Top. Membr. Transp. 5,51-136. 7. Boos, W. (1974) Annu. Rev. Biochem. 43,123-145. 8. Robbins, A. R. & Rotman, B. (1975) Proc. Nati. Acad. Sci. USA 72,423-427. 9. Ames, G. F. & Lever, J. (1970) Proc. Nati. Acad. Sct. USA 66, 1096-1103. 10. Ames, G. F. & Spudich, E. N. (1976) Proc. Natl. Acad. Sci. USA 73, 1877-1881. 11. Rhoads, D. B., Waters, F. B. & Epstein, W. (1976) J. Gen. Physiol. 67,325-341. 12. Rhoads, D. B. & Epstein, W. (1977) J. Btol. Chem. 252, 13941401. 13. Epstein, W. & Davies, M. (1970) J. Bacteriol. 101, 836-843. 14. Rhoads, D. B., Laimins, L. & Epstein, W. (1978), J. Bacteriol, in press. 15. Epstein, W. & Kim, B. S. (1971) J. Bacteriol. 108,639-644.
16. Ptashne, M. (1967) Proc. Nati. Acad. Sci. USA 57,306-400.
17. McEntee, K., Hesse, J. E. & Epstein, W. (1976) Proc. Nati. Acad. Sci. USA 73,3979-3983. 18. O'Farrell, P. Z.; Gold, L. M. & Huang, W. M. (1973) J. Biol. Chem. 248, 5494-5501. 19. Ptashne, M. (1970) Cold Spring Harbor Symp. Quant. Btol. 35,
283-294. 20. Ito, K., Sato, T. & Yura, T. (1977) Cell 11, 551-59. 21. Osborn, M. J. & Munson, R. (1974) in Methods in Enzymology, eds. Fleischer, S. & Packard, L. (Academic, New York), Vol. 31, Part A, pp. 642-653. 22. Lugtenberg, B., Meijers, J., Peters, R., Van der Hoek, P. & van Alphen, L. (1975) FEBS Lett. 58,254-258. 23. Boos, W., Hartig-Beecken, I. & Altendorf, K. (1977) Eur. J. Biochem. 72, 571-581. 24. Boos, W. & Gordon, A. S. (1971) J. Biol. Chem. 246,621-628. 25. Piperno, J. R. & Oxender, D. L. (1966) J. Biol. Chem. 241, 5732-5734. 26. Jean, D. H., Albers, R. W. & Koval, G. J. (1975) J. Biol. Chem. 250, 1035-1047. 27. Hopkins, B. E., Wagner, H. & Smith, T. W. (1976) J. Bid. Chem.
251,4365-4371.
28. Hokin, L. E. & Dahl, J. L. (1972) in Metabolic Transport, ed. Hokin, L. E. (Academic, New York), Vol. 6, pp. 2694308. 29. West, I. C. & Mitchell, P. (1974) Blochem. J. 144,87-90. 30. Post, R. L., Meritt, C. R., Kinsolving, C. R. & Albright, C. D.
(1960) 1. Biol. Chem. 235,1796-1802. 31. Kobayashi, H., Van Brunt, J. & Harold, F. M. (1978) J. B"o. Chem. 253, 2085-2092. 32. Postma, P. W. & Roseman4 S. (1976) Biochim. Bkoph. Acta 457,
213-257.
Biochemistry
Identification of the structural proteins of an ATP-driven potassium transport system in Escherichia coli (inner membrane proteins/Kdp transport system/kdp transducing phage/amber mutations/periplasmic protein)
LAIMONIs A. LAIMINS*, DAVID B. RHOADS*, KARLHEINZ ALTENDORFt*, AND WOLFGANG EPSTEIN*§ * Department of Biophysics and Theoretical Biology, § Department of Biochemistry, University of Chicago, Chicago, Illinois 60637; and t Institut fur Biologie, Universitit Tubingen, D 7400 Tfibingen 1, Germany
Communicated by Albert Dorfman, May 5, 1978
ABSTRACr The three structural proteins of the ATP-driven Kdp potassium transport system of Escherichia coli [Rhoads, D. B., Waters, F. B. & Epstein, W. (1976) J. Gen. PhysioL 67, 325-341] have been identified and found to be located in the inner membrane. The high-affinity repressible Kdp system is one of four potassium transport systems in E. coli. The Kdp proteins were identified both in growing cells as well as in heavily UV-irradiated cells infected with transducing phages carrying the kdp operon. Although all previously identified ATP-driven transport systems of Gram-negative bacteria have been shown to contain a periplasmic protein component, no evidence was found for such a component or for an outer membrane component of the Kdp system. The molecular weights of the three inner membrane proteins, KdpA, KdpB, and KdpC, were determined to be 47,000, 90,000, and 22,000, respectively. ATP or another high-energy phosphate compound serves as the energy source for a number of active transport systems in bacteria (1, 2). The characterization of ATP-driven systems in bacteria remains incomplete. Many of these transport systems in the enteric bacteria have a soluble, periplasmic "binding" protein that binds the substrate with high specificity and affinity (3-6). Such binding is believed to be the first step in transport (7) and is followed by translocation across the inner bacterial membrane by one or more proteins specific to the transport system. Although these additional proteins, presumably membrane proteins, have been genetically defined for several systems (8-10), they have not been biochemically identified for any bacterial system. Escherichia coli has a high-affinity repressible transport system for potassium, designated the Kdp system (11), which is ATP-driven (12). The function of this system depends on the expression of the four clustered kdp genes (13) comprising the kdpD regulatory gene and the kdpABC operon (14). Here we report the identification of the three proteins coded by the kdpABC operon. All are inner membrane proteins. The Kdp system does not appear to have a periplasmic protein component.
is dependent on the Kdp system up to an external K+ concentration of approximately 20 mM (11). The Kdp transducing phage, Xpkdp-1, has been reported to carry all three structural genes of the kdpABC operon (14). All transducing phages carry the XcI857 mutation. Phages carrying kdp amber mutations were prepared by lysogenizing a host carrying the desired mutation with Xpkdp-1, inducing the prophage by shifting the culture to growth at 420, and screening for Kdp- phages by the ML plate method (14). The genotype of the mutant phages was confirmed by their ability to complement a nonsupressible kdp point mutation in the same gene in a suppressing host but not in a nonsuppressing host. Growth Media and Conditions. Cells were grown with shaking at 370 in medium containing glucose (4 g/liter) as carbon and energy source. The phosphate-buffered media, referred to by K+ concentration in mM, have been described (15). Growth in K15 fully represses the Kdp system except in constitutive strains TL1000 and TL1007 which express the Kdp system at 12% of the fully derepressed rate due to the presence of the kdpDl7 mutation. All TK and TL strains express the Kdp proteins at a high rate when shifted from K15 medium to K5 or K10 medium, whereas wild-type strain Ymel expresses the Kdp system at a high rate only when grown in media containing less than 100 IAM K+. Labeling of Phage-Coded Proteins. Labeling was per-
formed by the method of Ptashne (16), with the modification of McEntee et al. (17). The cells were grown and irradiated in KLlS medium. After a 60-min recovery in K15 medium, the cells were infected and labeled for 60 min at 370 in either K10 or K115 medium, as indicated, with 10 ;tCi of "4C-labeled amino acid mixture (Schwarz/Mann) per 109 cells. Labeling Growing Cells. After derepression of the Kdp system by a shift from K1 15 medium to K5 medium, the cells at a concentration of 5 X 108/ml were labeled with 14C(labeled (0.3 ,Ci/ml) amino acid mixture (Schwarz/Mann). Labeling occurred over the interval from 15 to 135 min after transfer to K5.
Analysis of Labeled Proteins. Sodium dodecyl sulfate/ polyacrylamide electrophoresis and photofluorogram analysis were performed as described (17).
MATERIALS AND METHODS Bacteriophages and Strains. The strains used are listed in Table 1. In addition to the Kdp system, three other K+-transport systems are genetically defined in E. coli. In all TK and TL strains shown in Table 1, the function of the other two saturable K+-transport systems, TrkA and TrkD (11), is abolished by mutations (15). This facilitates genetic and transport studies because, in trkA trkD mutants, both growth and K+ transport
RESULTS Identification of Xpkdp-1 Coded Proteins. Genes introduced by a transducing bacteriophage are preferentially expressed when a host defective in DNA repair is irradiated with a high dose of UV, followed by phage infection. If, in addition, the host is lysogenic for Xind-, a noninducible mutant of X,
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
* Present address: Lehrstuhl fur Biochemie der Pflanzen, Ruhr-Universitat Bochum, D 4630 Bochum 1, Germany.
3216
F~
Biochemistry:
Laimins et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Bacterial strains* and bacteriophages Designation TL1000 TL1007 TL1011 TK2104/4 TK2121/21 TK2130/30 TK2131/31 Ymel xpkdp-l Xpkdp-l-A21 Xpkdp-1-B30 Xpkdp-l-C31
B
Pertinent genotypet
kdpDl 7 uvrA (Xind -) kdpDl 7 uvrA supD (Xind) kdpDll uvrA (Xind-) kdpA4 nadA with F100-kdpA4 kdpA21 nadA with F100-kdpA21 kdpB30 nadA with F100-kdpB30 kdpC31 nadA with F100-kdpC31
A
_
kdp+
c
Xc1857 kdpABC+ Xc 1857 kdpA21 Xc1857 kdpB30 Xc1857 kdpC31
* The TL and TK strains are also trkA401
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g c f e d b FIG. 2. Identification of the genes coding for the Kdp proteins. Labeling of proteins and electrophoresis were performed as for lane c of Fig. 1; a photofluorogram of the gel is shown. Nonsuppressing strain TL1000 was infected with Xpkdp-1 carrying the indicated amber kdp mutations: lane a, kdpA21; lanes b and e, kdpB30; lanes c and g, kdpC31. To show restoration of the missing band by an amber suppressor, supD strain TL1007 was infected with Xpkdp-1 carrying: lane d, kdpB30; lane f, kdpC31. a
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t The listed kdp alleles have been characterized as follows: kdpA4,
missense; kdpA21, amber; kdpB30, amber; kdpC31, amber; kdpDll, missense; kdpD17, missense resulting in partial constitutivity of the Kdp system.
expression of all X genes under control of X repressor is blocked. Under these conditions, bacterial genes carried by the transducing phage and not under repressor control, as well as the X repressor gene itself, are preferentially expressed. By using this protocol, the bacterial proteins coded by Xpkdp-l were iden-
tified (Fig. 1). When protein labeling was performed in K10 medium to derepress the kdp genes on the phage, four prominently labeled proteins were seen (Fig. 1, lane c). Three of these proteins, marked A, B, and C, were much less prominent when labeling was performed in K115 medium (Fig. 1, lane b) which, in this partially constitutive host, reduces expression of the kdp genes to 12% of the maximum rate. The three marked proteins were not seen in the uninfected control (Fig. 1, lane a), nor were they found after infection with wild-type X. These results suggest that the proteins marked A, B, and C are the products of the three genes of the kdpABC operon. Infection of host
I,
3217
B
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c a b FIG. 1. Identification of the Kdp proteins. Proteins synthesized after infection of UV-irradiated strain TL1000 with Xpkdp-1 were labeled with radioactive amino acids and separated electrophoretically on a gel containing 20% acrylamide and 0.07% methylene bisacrylamide. A photofluorogram of the gel is shown; migration is from top to bottom. Each sample contained 50,000 cpm. (a) Uninfected cells, labeled in K115 medium; (b) Xpkdp-l-infected cells, labeled in K115 medium; (c) Xpkdp-l-infected cells, labeled in K10 medium. Letters at the right identify the Kdp proteins and the X repressor.
strain TL1011, whose kdpDll mutation prevents expression of the kdp operon (13, 14), did not result in synthesis of proteins A, B, and C, demonstrating that synthesis of these proteins is under kdpD control. The fourth protein was identified as the X repressor. It was made after infection with wild-type X (data not shown) and its molecular weight of 27,000, estimated by using T4 proteins as standards (18), is consistent with that of the X repressor (19). The genes coding for each of the proteins were identified by examination of the proteins made after infection with mutant Xpkdp-1 phages carrying kdp amber mutations. In each infection, one of the three proteins was not synthesized (Fig. 2, lanes a-c). To make sure that absence of a protein was not due to some artifact, we examined proteins synthesized upon infection of hosts carrying an amber suppressor mutation with the kdp amber phages. The reappearance of the proteins in these hosts (Fig. 2, lanes d and f) confirmed that we were observing an effect due to chain termination by the amber mutations. The data show that kdpB codes for the largest protein (designated B), kdpA codes for the protein of intermediate size (marked A), and protein C (with the lowest monomer molecular weight) is coded for by kdpC. Identification in Growing Cells. The same three Kdp proteins were synthesized at a high rate in cells derepressed for the Kdp system. Membrane proteins of kdp diploid strains labeled under Kdp derepressing conditions are shown in Fig. 3. All three Kdp proteins were synthesized in the kdpA4 missense mutant strain, shown in lane g. The protein product of a particular gene is absent in the strain carrying an amber mutation in that gene (lanes d-f). We attribute the lack of a visible KdpC protein and the much decreased amounts of the KdpB protein in the strain with the kdpA21 amber mutation (Fig. 3, lane f) to a polar effect. The kdp operon is transcribed from kdpA to kdpC (14), so that a chain-terminating mutation near the promoter end of the kdpA gene will be polar on expression of kdpB and kdpC. The relatively small amounts of the KdpB and KdpC proteins made after infection with the kdpA21 phage (Fig. 2, lane a) suggest that this mutation is also polar when the kdp operon is expressed in a heavily UV-irradiated host. All three Kdp proteins were stable; there was no apparent loss of label from any of the three proteins during a 2-hr chase in
3218
Biochemistry: Laimins et al. awk4w
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 3. Identification of the Kdp proteins among membrane proteins of growing cells. Strains examined were homozygous kdp diploids carrying kdp point mutations. The separating gel contained 15% acrylamide and 0.4% methylene bisacrylamide. Membrane proteins of the kdpA4 mutant strain: lane a, cells labeled under Kdp repressing conditions; lane b, cells labeled under Kdp derepressing conditions and subjected to a 2-hr chase in nonradioactive medium during growth under Kdp repressing conditions; lane c, outer membrane fraction of cells labeled under Kdp derepressing conditions and separated as described by Ito et al. (20). Membrane proteins labeled under Kdp derepressing conditions in strain with indicated mutation: lane d, kdpC31; lane e, kdpB30; lane f, kdpA21; lane g, kdpA4.
which the cells were grown under repessing conditions after labeling (Fig. 3, lane b). A control showing that none of the Kdp proteins was synthesized under repressing conditions is also included (lane a). The molecular weights of the Kdp proteins were estimated by comparing their mobilities in sodium dodecyl sulfate/ polyacrylamide gels with those of protein standards in two similar experiments (Fig. 4). Comparative plots of the logarithm of molecular weight as a function of migration distance gives values of 85,000 and 95,000 for KdpB, 47,000 and 48,000 for KdpA, and 22,000 and 23,000 for KdpC. Values obtained in a similar experiment for Xpkdp-1 coded Kdp proteins using T4 early proteins (18) as standards led to estimates of 96,000, 46,000, and 21,000. Membrane Association. All three proteins of the kdpABC operon were firmly associated with the inner membrane of E. coli. Examination of separated inner and outer membranes from a wild-type strain grown either under Kdp repressing or derepressing conditions showed that all three proteins were in the inner membrane (Fig. 5). There was no trace of any of these proteins in the outer membrane fractions shown in this experiment or in the outer membrane fraction of mutant kdpA4 (Fig. 3, lane c). We found no evidence for a periplasmic protein component of the Kdp system, despite considerable effort to identify such a component. Neither one- nor two-dimensional gel electrophoretic analysis (23) of cold osmotic shock-releasable proteins (24) revealed any protein whose behavior was consistent with that of a protein coded for by or regulated in parallel with the
2
6 4 Relative migration distance
8
10
FIG. 4. Determination of the monomer molecular weight of the Kdp proteins. Shown are the results of two experiments in which migration of the Kdp proteins and of protein standards were compared. Protein standards and radioactively labeled Kdp proteins made in a diploid kdpA4 mutant were separated in the presence of sodium dodecyl sulfate as in the experiment of Fig. 3. The standards were detected by staining with Coomassie blue and the Kdp proteins, by fluorography. Protein standards and their molecular weights (X10-3) were: phosphorylase B, 92; bovine serum albumin, 67; glutamate dehydrogenase, 53; creatine phosphokinase, 40.5; carbonic anhydrase, 30; soybean trypsin inhibitor, 21.5; f3-lactoglobulin, 18.3; lysozyme, 14.4. The estimates are 95,000, 47,000, and 23,000 from the curve at the left and 85,000, 48,000, and 22,000 from that on the right for the B, A, and C proteins, respectively.
kdp genes. The stability of the Kdp inner membrane proteins in a chase experiment (Fig. 3, lane b) rules out the possibility that any serve as precursors of soluble proteins. Similar chase experiments with Xpkdp-l-coded proteins showed no change in membrane association or size during a 12-hr chase in nonradioactive medium. DISCUSSION All three structural proteins of the Kdp transport system are here shown to be stable inner membrane proteins. No periplasmic protein or outer membrane protein component was found for the Kdp system. The approximate monomer molecular weights of these proteins are 47,000, 90,000, and 22,000 for the KdpA, KdpB, and KdpC proteins, respectively. Because
A
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0
FIG. 5. The Kdp proteins are located in the inner membrane. Strain Ymel was grown in K5 medium to repress or in KO.05 medium to derepress the Kdp system. Isolated inner and outer membrane fractions from each of these cultures were obtained by the method of Osborn et al. (21), subjected to electrophoresis (22), and stained with Coomassie blue. Two amounts of each fraction containing approximately 50 and 80 Ag of protein were analyzed: lanes a and c, inner membranes, Kdp derepressed; lanes b and d, inner membrane, Kdp repressed; lanes e and g, outer membrane, Kdp derepressed; lanes f and h, outer membrane, Kdp repressed.
Biochemistry:
Proc. Natl. Acad. Sci. USA 75 (1978)
Laimins et al.
other ATP-driven transport systems in enteric bacteria have a periplasmic protein (3-6), we made an extensive search for such a component of the Kdp system. Evidence reported earlier suggesting a periplasmic protein component included (i) a moderate loss (30%) of Kdp transport activity after osmotic shock, and (ii) a protein released by cold osmotic shock apparently dependent on the kdp genotype of the strain (12). The studies presented above show the previous suggestion to be incorrect. Gel electrophoretic analysis of proteins released by cold osmotic shock from mutant and wild-type cells grown under different conditions demonstrate that no periplasmic protein is coded by the kdpABC operon or is regulated in parallel with the Kdp system. The reduction in Kdp transport activity after osmotic shock is most likely a nonspecific effect as noted by others in systems that do not have periplasmic proteins (25). Periplasmic proteins function as the initial substrate-binding component of other ATP-driven transport systems. Because the Kdp system does not appear to have such a protein, initial substrate binding must occur on the inner membrane. We have sought genetic evidence on the binding site by isolating mutants with an altered Km for K+. Preliminary studies of such mutants suggest that the 47,000 molecular weight KdpA protein represents the substrate binding site. The Kdp system resembles the Na+, K+-ATPase that transports these two cations across the plasma membrane of animal cells. The two subunits of this ATPase have monomer molecular weights of approximately 95,000 and 60,000 (26, 27), similar to the sizes of the KdpB and KdpA proteins, respectively. There are some striking differences as well: (i) The Kdp system has a third small subunit, the KdpC protein. (ii) The Kdp system does not appear to perform K+ uptake coupled to Na+ extrusion characteristic of the animal cell system (28). The rate of K+ uptake by bacteria suspended in buffer containing less than 20 ,uM Na+ is similar to that of cells in 100 mM Na+ buffer (12). Bacteria use a separate system to pump Na+ out, the proton Na+ antiporter (29), rather than a single system, as animal cells do, for both transport functions. The Kdp system may couple K+ uptake to extrusion of another cation such as a proton; alternatively, it may be an electrogenic process that depends on other ionic movements to maintain charge balance. (iii) The Km of the Kdp system is 2 ,gM, almost 3 orders of magnitude lower than the Km of the Na+,K+-ATPase for K+ (30). Accompanying this low Km is the ability to maintain the very high transmembrane concentration ratio for K+ of 4,000,000:1 (11). (iv) The Kdp system is a repressible transport system, made only when the other transport systems are unable to meet the cell's K+ needs. In contrast, the Na+,K+-ATPase is always needed to maintain the required cation content of animal cells. The identification of the Kdp proteins makes this system amenable to biochemical investigation. Because all identified components of the Kdp system are membrane bound, we expect this system to resemble the transport ATPases of animal cells in requiring only membrane-bound components to perform substrate translocation and energy coupling. Kobayashi and coworkers (31) have recently described an ATP-driven Ca2+ transport system from Streptococcus fecalts. Because this system is active in vesicles, it must require only membrane-bound components. The only well-characterized bacterial transport system in which high-energy phosphate bond energy is used, the phosphoenolpyruvate-dependent phosphotransferase system, requires two soluble proteins in addition to membrane
3219
components to perform transport energized by phosphoenolpyruvate (32). We anticipate that the ability to perform a full range of genetic manipulations, in addition to applying biochemical techniques, will be a significant advantage in elucidating the molecular mechanism of K+ transport by this system. The authors thank B. Ewersmeyer-Wenk, G. Heinze.lmann, and J. E. Hesse for excellent technical assistance, K. McEntee for advice in experiments with Xpkdp-l, and W. Boos for advice on two-dimensional gel electrophoresis. This work was supported by financial assistance from Prof. Dr. V. Braun, by traineeship awards to L.A.L. and D.B.R. from National Institutes of Health Training Grant GM780, and by the following research grants: PCM75-14016 from the National Science Foundation, GM22323 from the National Institutes of Health, and SFB 76 from the Deutsche Forschungsgemeinshaft. 1. Berger, A. E. (1973) Proc. Nati. Acad. Sci. USA 70, 15141518. 2. Simoni, R. D. & Postma, P. W. (1975) Annu. Rev. Biochem. 44, 523-551. 3. Cowell, J. L. (1974) J. Bacteriol. 120, 139-145. 4. Curtis, S. J. (1974) J. Bacteriol. 120, 295-303. 5. Berger, E. A. & Heppel, L. A. (1974) J. Biol. Chem. 249, 7747-7755. 6. Boos, W. (1974) Curr. Top. Membr. Transp. 5,51-136. 7. Boos, W. (1974) Annu. Rev. Biochem. 43,123-145. 8. Robbins, A. R. & Rotman, B. (1975) Proc. Nati. Acad. Sci. USA 72,423-427. 9. Ames, G. F. & Lever, J. (1970) Proc. Nati. Acad. Sct. USA 66, 1096-1103. 10. Ames, G. F. & Spudich, E. N. (1976) Proc. Natl. Acad. Sci. USA 73, 1877-1881. 11. Rhoads, D. B., Waters, F. B. & Epstein, W. (1976) J. Gen. Physiol. 67,325-341. 12. Rhoads, D. B. & Epstein, W. (1977) J. Btol. Chem. 252, 13941401. 13. Epstein, W. & Davies, M. (1970) J. Bacteriol. 101, 836-843. 14. Rhoads, D. B., Laimins, L. & Epstein, W. (1978), J. Bacteriol, in press. 15. Epstein, W. & Kim, B. S. (1971) J. Bacteriol. 108,639-644.
16. Ptashne, M. (1967) Proc. Nati. Acad. Sci. USA 57,306-400.
17. McEntee, K., Hesse, J. E. & Epstein, W. (1976) Proc. Nati. Acad. Sci. USA 73,3979-3983. 18. O'Farrell, P. Z.; Gold, L. M. & Huang, W. M. (1973) J. Biol. Chem. 248, 5494-5501. 19. Ptashne, M. (1970) Cold Spring Harbor Symp. Quant. Btol. 35,
283-294. 20. Ito, K., Sato, T. & Yura, T. (1977) Cell 11, 551-59. 21. Osborn, M. J. & Munson, R. (1974) in Methods in Enzymology, eds. Fleischer, S. & Packard, L. (Academic, New York), Vol. 31, Part A, pp. 642-653. 22. Lugtenberg, B., Meijers, J., Peters, R., Van der Hoek, P. & van Alphen, L. (1975) FEBS Lett. 58,254-258. 23. Boos, W., Hartig-Beecken, I. & Altendorf, K. (1977) Eur. J. Biochem. 72, 571-581. 24. Boos, W. & Gordon, A. S. (1971) J. Biol. Chem. 246,621-628. 25. Piperno, J. R. & Oxender, D. L. (1966) J. Biol. Chem. 241, 5732-5734. 26. Jean, D. H., Albers, R. W. & Koval, G. J. (1975) J. Biol. Chem. 250, 1035-1047. 27. Hopkins, B. E., Wagner, H. & Smith, T. W. (1976) J. Bid. Chem.
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28. Hokin, L. E. & Dahl, J. L. (1972) in Metabolic Transport, ed. Hokin, L. E. (Academic, New York), Vol. 6, pp. 2694308. 29. West, I. C. & Mitchell, P. (1974) Blochem. J. 144,87-90. 30. Post, R. L., Meritt, C. R., Kinsolving, C. R. & Albright, C. D.
(1960) 1. Biol. Chem. 235,1796-1802. 31. Kobayashi, H., Van Brunt, J. & Harold, F. M. (1978) J. B"o. Chem. 253, 2085-2092. 32. Postma, P. W. & Roseman4 S. (1976) Biochim. Bkoph. Acta 457,
213-257.
