The KDP ATPase of Escherichia COP KARLHEINZ ALTENDORF? ANNETTE SIEBERS?= AND WOLFGANG EPSTEINd>e bDepartment of Microbiology University of Osnabmeck 4500 Osnabmeck, Germany dDepartmentof Molecular Genetics and Cell Biology University of Chicago Chicago, Illinois 60637 Potassium transport across the cytoplasmic membrane of Escherichia coli is a complex process involving multiple and separate systems for influx and efflux. (See reviews by Walderhaug et a1.l and Bakker?) The most completely characterized system is Kdp, a P-type transport ATPase that serves to accumulate K+ when the external concentration of this ion is Kdp is not essential; the only phenotype of mutants lacking Kdp is failure to grow at low K+ concentrations. The ka'pFABC (formerly M p M C ) operon encodes the three proteins, KdpA, KdpB, and KdpC, identified as part of the Kdp complex and the small hydrophobic KdpF peptide of unknown function. Kdp expression appears to be determined by turgor pressure, mediated by the products5 of the adjacent ka'pDE operon that encodes a pair of typical sensor kinase-response regulator proteins.6 STRUCTURE AND FUNCTION OF Kdp In this section, models of the structure and function of the Kdp subunits and their integration in the KdpABC complex are presented based on: (1) experimental data on phosphorylation and topology described below, (2) predictions of hydrophobicity and secondary structure, from the sequence of the proteins based on the nucleotide sequences of the kdpABC genes,7 and ( 3 ) comparisons based on homology of Kdp to other P-type ATPases.p-lo The KdpB Subunit

KdpB is the catalytic subunit. The model of KdpB (FIG. 1) is of an integral membrane protein with six membrane-spanning a-helices, a small and a large cytoplasmic protein domain, and little periplasmic exposure. This structure matches the key features of all P-type ATPases" and is compatible with the proposed nThis work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 171) and the Fonds der Chemischen Industrie to K. A,, and grant GM22323 from the National Institutes of Health to W. E. K. A. thanks the Volkswagenstiftungfor financial support for his stay at the University of Chicago. C PADDRESS: ~ Biotechnology ~ ~Laboratory, ~ Departments ~ of Microbiology and Biochemistry, University of British Columbia, Vancouver, B.C. V6T 123 Canada. =Addressfor correspondence:Dr. Wolfgang Epstein, University of Chicago-MGCB, 920 E. 58th St., Chicago, IL 60637-1474. 228

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headpiece-stalk model for this class of enzymes.8According to the three-dimensional image reconstruction of the CaZ+-ATPase,12the cytoplasmic domains constitute a large globular region connected by a narrow stalk to the hydrophobic membraneanchoring segment of the enzyme. In KdpB, the stalk would be formed by the five extramembranous regions immediately adjacent to the transmembrane helices 1,2, 3,4, and 5 (FIG. 1). Serrano8 has described 10 regions of homology that are common to all P-type ATPases (sequences a-j in FIG. 1). The only conserved region with hydrophobic character (region e) is located in transmembrane segment no. 4 adjacent to the catalytic site. A role for this region in energy transduction has been proposed.I3 Insights into the molecular mechanism of cation transport by P-type ATPases come from work on the yeast plasma membrane H+-ATPase8J4 and the CaZ+ATPase of sarcoplasmic reticulum. With the latter enzyme, the sites of phosphorylation,1°J5 of ATP binding1°J6 of cation binding,17J8and of conformational EIP-EzP transition^'^ have been investigated by site-directed mutagenesis. Apart from an approximate identification of the site of phosphorylation described below, direct experimental evidence about functional residues of the Kdp-ATPase is lacking. However, the many similarities between P-type ATPases allow an informed guess as to the Kdp counterparts of functional residues characterized in other members of the family. 1. The catalytic site. The phosphorylation site (P in FIG.1) has the consensus sequence DKTGT[L/I]T in all known P-type ATPases.IO The corresponding residue beginning this sequence in KdpB is Asp307. 2. The FITC binding site. The binding site for fluorescein isothiocyanate (FITC in FIG. 1) has been proposed to be part of or close to the ATP binding region, because all FITC-sensitive ATPases can be protected from this compound by ATP.zo,21A lysine has been identified as the modified residue. It is conserved in all eukaryotic P-type ATPases and in KdpB (Lys395) but not in the other prokaryotic cation pumps. Site-directed mutagenesis of the lysine in the FITC binding site of the Ca2+-ATPase did not support a direct involvement of this residue in ATP binding.Io 3. The ATP binding site. Taylor and Green22have proposed a detailed model of the spatial structure of the nucleotide binding domain of cation pumps, in which conserved amino acids are grouped around a postulated ATP binding cleft. Three conserved aspartic acid residues (corresponding to Asp447, Asp473, and Asp522 in KdpB; ATPI, ATP2 and ATP3 in FIG. 1) are located within the predicted ATP binding loops. Mutagenesis of the respective residues in the yeast H+-ATPase alters the nucleotide specificity of the enzymes, pointing to their participation in nucleotide binding.14 Two other putative ATP binding sites have been identified by the use of ATP analogs. Adenosine triphosphopyridoxal (AP3PL in FIG.1) binds to a lysine residue in the Ca2+-ATPaseof sarcoplasmic reticulum23that corresponds to Lys499 in KdpB, and the alkyiatingATP analogy [4-(N-2-chloroethyl-N-methylamino)]benzyIamide ATP (CIRATP in FIG.1) reacts with an aspartate residue in the Na+,K+A T P ~ that s ~corresponds ~ ~ to Asp518 in KdpB. 4. Conformational changes. The sequence motif Thr-Gly-Glu is located within the cytoplasmic domain between transmembrane segments 2 and 3 of P-type ATPases. Site-specificmutations in the Ca2+-ATPase have revealed that these three residues participate in the conformational change associated with the EIP-EZP transition (EIP c,E2P in FIG. l)." The corresponding motif in Kdp begins with Thr169. 5. Role in K+ binding. Site-directed mutagenesis in which Asp300 was replaced by Asn or Glu does not block activity, but results in a change in the K, for K+ of 380

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pM versus 12 pM for the ~ i l d - t y p eThese . ~ ~ data lend support to the notion that Asp300 is part of the regulatory site for K+ stimulation. Certain essential residues are conserved in some cation pumps but not in the Kdp-ATPase. These include (1) the glycine residue that determines the sensitivity of the Schizosaccharomyces pombe H+-ATPase to vanadate26and seems to be involved in the E I P to E2P conformational changes of the phosphate site in the Ca2+-ATPase2' (corresponding position in KdpB is shown by 1 in FIG.l ) , and (2) the lysine residue (2 in FIG. 1 ) that represents one of the binding sites for the ATP analog 5'-pfluorosulfonylbenzoyladenosine(FSBA) in the Na+,K+-ATPase28;this lysine residue is not conserved in any prokaryotic ATPase."'

The KdpA Subunit Thc KdpA subunit is a hydrophobic protein that is predicted to span the membrane with 12 a-helical segments (FIG.2). Genetic studies suggest that KdpA is

Cytoplasm

n n

V

W

Periplasm FIGURE 2. The predicted structure of the KdpA protein, showing the 12 predicted membranespanning segments and particular residues or sites discussed in the text. (Reproduced from ref. 4 with permission.)

involved in K+ binding and translocation." Twelve different kdpA mutants have been isolated with markedly reduced affinity for K+ but virtually unaltered maximum rates of transport (K,,, mutants). The mutations producing this change are clustered in three extramembrane loops, between transmembrane helices 2 and 3, between 4 and 5 , and between 10 and 11" (and unpublished observations). These mutations appear to identify a periplasmic K+ binding pocket. A fourth cluster of mutations, in residues 345-369, are in predicted transmembrane helices 7 and 8. This last cluster suggests the existence of intramembranous K+ binding sites that are in the path of transmembrane movement of K+. Acidic residues in the membrane that may b e part of this path are in helices 5,7, and 8 (Glu302 + Glu370 + Glu272, FIG.2). KdpA does not have significant sequence similarity with any other known protein. However, its overall architecture is similar to that established for several secondary membrane p o r t e r ~ . ~Inl . particular, ~~ the assembly from 12 membrane-

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spanning a-helices seems to be the structural paradigm of carriers that function in symport or antiport with H+ or Na+ such as those for sugars, citrate, or t e t r a c y ~ l i n e . ~ ~ There are interesting analogies of KdpA to the lactose carrier3' of E. coli beyond the unlfying 12-helix structural motif: the occurrence of 7-9 Pro residues, 3 Glu residues, and an excess of positive charge in the membrane part. Like most membrane proteins, the internally exposed regions have a positive net charge, whereas the external ones have a negative net The KdpC Subunit

KdpC (FIG.3) appears to contain only one membrane-spanning a-helix close to the NH2-terminus. The rest of the protein presumably extends into the cell interior, but as indicated below is not extensively exposed to the cytoplasm in the KdpABC complex. Further characteristics of the secondary structure are two antiparallel p-sheets and an amphipathic a-helix at the COOH-terminus. The predicted structure of KdpC is similar to that of the p1 and p2 isoforms of the small subunit of the N a + , K + - A v a ~and e ~ ~the p subunit of the H+,K+-ATPase,%in that all of these proteins have only one transmembrane helix near their NH2-termini that is followed by a large extramembranous domain. However, this resemblance is probably fortuitous, as the p subunits of the eukaryotic enzymes, in contrast to KdpC, have their large extramembranous part exposed externally where they are extensively glycosylated and there is only marginal sequence similarity. There may well be a similarity in function, because the p subunits have been implicated in the assembly of eukaryotic P-type ATPase~,3~ and as mentioned below KdpC may assist in assembly of the KdpABC complex. We recently obtained evidence for the existence of an ATP binding site on KdpC that may have a regulatory function. Photoaffinity labeling of the Kdp complex with [32P]-8-azido-ATPresulted in not only labeling of KdpB but even greater labeling of KdpC. Labeling was in the NH2-terminal cyanogen bromide fragment of KdpC, Metl-Met75 (S. Drose and K. Altendorf, unpublished observations). The notion that

COOH

FIGURE 3. The predicted structure of the KdpC protein as discussed in the text. Boxes with arrows represent regions of p-sheet structure, other boxes are regions of predicted a-helical structure, and filled-in boxes are predicted to be hydrophobic. The small arrows show predicted p turns. (Reproduced from ref. 4 with permission.)

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K+

FIGURE 4. The proposed structure of the KdpABC complex, shown as a dimer with KdpA subunits in contact while the KdpC subunits mediate interaction between KdpA and KdpB. Each KdpA subunit is shown as a separate path for K+ transport. (Reproduced from ref. 4 with permission.)

ATP may play a regulatory role in addition to that of substrate has arisen independently from enzymatic studies of the Kdp complex.” The KdpF Peptide The sequence of the kdp region’ indicated that there was an additional open reading frame, kdpF, just upstream of the kdpA gene and encoding a very hydrophobic 29 amino-acid ~ e p t i d e . ~A’ small peptide consistent with this size has been detected when the kdpFA5C operon is selectively transcribed (T. Moellenkamp and K. Altendorf, unpublished observations), and a protein fusion to lucZ made in vitro was found to produce a membrane-bound form of P-galactosidase (L. Covello and W. Epstein, unpublished observations) whose expression was like that of Kdp (see below). Thus, this small peptide is expressed, but its role remains undetermined.

The KdpABC Complex From genetic studies it appears that the KdpABC complex is oligomeric with respect to the KdpA p r ~ t e i n .Preliminary ~ data suggest that each of the three The picture subunits is present in equimolar amounts, as in a AzBzCz emerging for the Kdp complex is that of a membrane-embedded core of two KdpA molecules, surrounded by the intramembrane segments of KdpC and KdpB (FIG.4). As suggested for other P-type ATPases, a stalk segment extends from the membra-

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nous parts of KdpB to form a large globular region that covers most of the extramembranous parts of KdpC. In comparison with other P-type ATPases, Kdp is unique because it has both high affinity and high specificity for K+ as well as regulation of activity by turgor (see below). During evolution, a single type of subunit may not have sufficed to reach this degree of specialization. Rather, the precursor of KdpB may have specialized in ATP hydrolysis and delegated transport to the precursor of KdpA which originated from the family of secondary ion porter^.^^,^^ Evidence that KdpB has abandoned transport comes from examination of the sequences between membrane-spanning regions 1 and 2 and between 3 and 4 of KdpB (residues 54-62, and 239-246, respectively). These regions are short and not very polar, having at most one strongly polar residue," unlike all other P-type ATPases, where these small regions have at least two and often many more highly polar residues. The relatively apolar nature of these loops in KdpB suggests that they serve largely as membrane anchors and interact with membrane portions of other subunits but do not extend into the periplasmic space outside. If they do not extend into the periplasmic space, they probably do not participate directly in transmembrane ion movement. The postulated division of labor between KdpB and KdpA may have necessitated a third component, KdpC, to assemble the other two and mediate their interactions. Specifically, KdpC may transmit conformational changes between the energyproducing KdpB subunit and the energy-consuming and K+-transporting KdpA subunit. Experimental evidence for an integrative function of KdpC comes from studies with kdpB and kdpC amber mutants. Solubilization and purification experiments revealed that KdpB and KdpA are not associated in the absence of KdpC; by contrast, a stable complex of KdpA and KdpC is formed in the absence of KdpB (A. Siebers, W. Epstein, and K. Altendorf, unpublished observations). In the Ca2+-ATPaseof the sarcoplasmic reticulum the high affinity binding sites for Ca2+ ions are localized within the transmembrane domain.17J8 These binding sites involve four negatively charged and two polar residues, 3 Glu, 1 Asp, 1Am, and 1Thr. Alignment of P-type ATPases shows a high degree of conservation of these six residues in all eukaryotic P-type ATPases as well as in the prokaryotic MgtBA T p a ~ e . 3According ~ to our model of KdpB (FIG.l), four of the six analogous residues are located outside the membrane. The only acceptable membraneintegrated counterpart in KdpB would be Asp673 (analogous to a Glu residue in the Ca2+-ATPase).As noted above, the KdpA subunit possesses three Glu residues in its membranous parts that may constitute an ion pathway (FIG.2).

Purification

The original protocol for the isolation of the Kdp complex was based on solubilization by the detergent Aminoxid WS-35 (C7-17-l-alkoylamino-3-dimethylaminopropane-3-N-oxide) from membranes of Kdp-induced cells followed by several chromatographic step^.^^,^^ The resulting preparation was over 90% pure, but due to marked sensitivity of the KdpB subunit to proteolytic attack, it had a relatively low specific activity of about 700 kmol g-' min-I and a modest activity yield (6.5%). Recently the purification has been simplified to a two-step procedure involving ion-exchange and dye-ligand chromatography, yielding enzyme of higher purity and activity.4l The purified native Kdp complex and each of its individual sodium-dodecyl sulfate-denatured subunits have been used to raise monospecific polyclonal antibodies?'

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Phosphorylated Intermediate Kdp forms a phosphorylated intermediate of KdpB36 whose properties are similar to those of other ATPases of this group. The intermediate is acid stable and labile to alkali and to hydroxylamine, properties characteristic of acyl phosphates. Experiments to identify the phosphorylated residue are in progress. By protein microsequencing of KdpB-derived cyanogen bromide fragments, the phosphorylation site has been localized to the 100 amino acids between Leu283 and Met383 (A. Siebers, R. Broermann, and K. Altendorf, unpublished observations). This agrees with sequence comparisons that predict Asp307 to be the site of p h o s p h ~ r y l a t i o n . ~ ~ ~ Other data suggestive of an essential catalytic function of Asp307 come from site-directed mutagenesis? replacing Asp307 by any of a number of other residues abolishes both K+ transport activity and phosphoenzyme formation.

[ll

Ei+ATP

EI-ATP

+

E2.K

El-P + A D P

f

FIGURE 5. A proposed reaction scheme of phosphorylation by Kdp. Steps 1, 2, and 4 are very similar to those of the Na+,K+-ATPaseexcept no internal substrate is shown as none is known. Step 3 is shown in two different versions. Enclosed in a dashed box and labeled A is the typical scheme for eukaryotic Kf-transporting enzymes in which K+ loading releases phosphate from E2-P. The alternative suggested for Kdp and discussed in the text separates the rupture, in step 3a, of the covalent linkage of P to Ez in E2-P from the K+ loading step, 3b.

Kinetic studies have shown that P-type ATPases participate in a cycle involving phosphorylation and dephosphorylation and ion binding, occlusion, and release. A 'high-energy' form, El, as well as a 'low-energy' form, E2,which exist in phosphorylated as well as dephosphorylated forms, have been characterized (reviewed by GIynn and K a r l i ~ h ~The ~ ) . two phosphorylated forms have an identical covalent structure, indicating the differences are conformational. The suggested reaction scheme for Kdp of FIGURE5 is slightly modified from that for other enzymes of this type by the inclusion of an alternate to step 3, the conversion of E2-P to E2 with bound K+. The diagram implies that the only cation moved is K+, but coupled movement of another ion by Kdp has not been totally excluded. If Kdp did not transport any cation out, it would be the only P-type ATPase that did not do so; all whose stoichiornetry have been examined appear to transport some cation out and at least one group, the H+-ATPases of fungi, export H+ out but bring in no counter ion.x Recent transport studies in vesicles mentioned below have given no evidence for coupled proton movement. Movement of Na+ by Kdp is considered implausible,

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given the absence of any Na+ requirement for Kdp transport activity in vivo or for Kdp ATPase activity in vitro. The reaction cycle of Kdp resembles that of other P-type ATPases. Kinetic analysis using a highly purified enzyme43indicates very rapid formation of phosphoenzyme at concentrations of ATP as low as 1 pM.A high rate of ADP-ATP exchange and an even more rapid discharge by ADP supports the presence of an El-P form of intermediate, of high energy as it is in equilibrium with ATP. The much higher affinity for ATP in the formation of phosphoenzyme than for ATPase activity36leads us to include step 4 in which ATP binds to the Ez.K+ complex at low affinity to liberate K+ inside and form the El .ATP complex to begin the cycle again. Two properties of the phosphorylated intermediate of Kdp are atypical; over 96% of the phosphorylated intermediate is rapidly discharged by ADP, typical of El-P, and none appears to be discharged by K+, considered characteristic of E2-P (for enzymes that transport K+). When further formation of phosphorylated intermediate is blocked by an excess of cold ATP, the rate of loss of phosphorylated intermediate is the same whether K+ is present or not." Failure to detect an E2-P form of the intermediate might lead one to conclude that the El-P to Ez-P conversion is slow and rate limiting. However, K+ stimulates ATPase activity four- to fivefold, indicating that a K+-dependent step is rate limiting. We explain this result by dividing step 3 into two parts: in step 3a there is spontaneous dephosphorylation of E2-Pthat does not involve K+,yielding a form of E2 with high affinity for K+,while in step 3b K+ is bound (occluded) to yield the substrate for binding of ATP in step 4. The product of step 3a is shown as free EZin FIGURE5, but it might retain phosphate in a bound form and so resemble the low energy E2-P form. If this form has bound phosphate, it would be released in step 3b. These kinetic data need to be interpreted in light of two other properties of the enzyme. The in vitro activity of Kdp is low, in the range of 1.1-1.4 mmol g-'min-' in the best purified preparations. If the active enzyme is a dimer of 300,000 daltons, this activity implies a turnover rate of only 5/sec, far below that of other P-type ATPases. The low rate is observed in crude membranes as well, where the in vitro hydrolysis rate is about an order of magnitude lower than that expected based on transport rates in vivo and energetic considerations.44In vitro there is also considerable activity in the absence of K+, typically 20-25% of the rate with saturating K+, and this is true of crude membranes as well as purified preparations. This uncoupled activity may be an artifact of the in vitro system or it might be inherent in Kdp with the same rate in vivo and in vitro. If so, it would represent only about 2% of the full rate in vivo, as the latter is low for some reason. We postulate that some factor lost in the process of cell disruption, probably a soluble factor, is needed for normal activity of Kdp. This factor would be needed for optimal interactions between the KdpA subunit that binds K+ and the KdpB subunit that provides phosphate bond energy. Poor interaction could account for a low rate of step 2 which presumably represents a conformational change in KdpA (as well as KdpB) and/or a low rate of step 4 which also involves conformational changes in both K+-binding and ATP-binding subunits. The existence of such a factor is purely hypothetical, as no evidence in support of it has been obtained. TOPorogv

The topology of the Kdp proteins has been investigated in membrane vesicles of defined orientation that were exposed to membrane-impermeable reagents such as pro tease^^^*^^ or the protein-modifying reagent [l~I]diazo-iodosulfanilicacid.2g From

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these studies, KdpB appears to be exposed for chemical modification and proteolytic digestion from both sides of the membrane, although much more rapidly digested from the cytoplasmic side. KdpA is almost resistant to proteases and does not react with the surface label from either side. The results with KdpC are contradictory: this subunit was not iodinated from either side but could be digested from the cytoplasmic side after prolonged incubation. We interpret these data to suggest that KdpC is shielded by the cytoplasmic parts of KdpB (FIG.4). This protection of KdpC is effective against surface labeling but is impaired after the protective region of KdpB is cleaved by the p r ~ t e a s e . ~ ~

Kdp Transport in Vesicles

To examine transport in right-side-out vesicles, ATP must be provided inside. We have done this by using strains carrying plasmid pBR322-pgt2 coding for the phosphoglycerate uptake system of Salmonella typhirnuri~m.~~ This transport system also accepts phosphoenolpyruvate (PEP) as a substrate. In this way, PEP taken up from the medium together with pyruvate kinase, ADP, and inorganic phosphate, provided to the intravesicular space during lysis of the spheroplasts, constitute an ATP-generating system. This system was recently successful in demonstrating uptake of K+ by the Kdp system to attain a 55-fold concentration gradient of K+ across the vesicle membrane (R. Kollman and K. Altendorf, unpublished observations). Uptake of K+ is inhibited by bafilomycin B1,a well-known inhibitor of P-type ATPases. However, we have not detected movement of H+,in symport or in antiport with K+. This negative result leads us to suggest that uptake of K+ via Kdp may be an electrogenic process. The reconstitution of the purified Kdp complex into liposomes has been only partly successful. About 50% of the starting ATPase activity has been found associated with the liposomes. The orientation of the Kdp complex seems to be random, as t h e addition of an excess of detergent leads to a doubling of K+stimulated ATPase activity as would be expected if only half of the ATP-binding sites were exposed externally. We have not so far been successful in demonstrating efflux of K+ from K+-loaded proteoliposomes upon the addition of ATP to the medium (R. Kollmann and K. Altendorf, unpublished observations).

REGULATION OF Kdp

The activity of Kdp is subject to controls on both the activity of the system when present and the expression of the system. Both of these types of control help Kdp in its physiological role as an additional system required only when external K+ is low. Cytoplasmic K+ is the major cationic osmotic solute47in E. coli, serving to maintain an acceptable level of turgor pressure, the difference between external and internal osmotic pressure characteristic of cells with walls. We can thus view regulation of Kdp as subordinated to the need of the cell to maintain turgor pressure. In view of the fact that Kdp activity in vesicles was only recently demonstrated as noted above, our knowledge of regulation is based on in vivo measurements. To avoid potential confusion that could result from activity of other K+ transport system, these studies were done in mutants lacking Kup (formerly TrkD) and Trk (formerly TrkA), so that over 98% of the residual activity is due to Kdp.48 The most convenient measurement, and the one that gives the highest rates, is net uptake in cells depleted

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of K+ where rates in the range of 100-150 Fmol g(dry wt)-l min-l are obtained. It is this data that suggests the in vivo rate of purified Kdp ATPase should be at least 10 mmol g-lmin-l, as in these strains Kdp proteins represent at most 2% of membrane proteins, the latter represent about 15% of total proteins, and energetic considerations4 suggest that no more than 2 K+ ions are transported per molecule of ATP hydrolyzed. Control of Kdp Activity

The high rate of uptake in K+-depleted cells is only an initial rate. Once cells restore their desired pools of K+ and turgor, they gradually reduce influx as they return to the steady-state K+ exchange characteristic of the medium. The factors that stimulate uptake in depleted cells or conversely that inhibit uptake in K+-replete cells have not been characterized. The high internal Na+ and presumed low internal pH of K+ depleted cells48could well stimulate uptake, while high internal K+ may inhibit. One factor that can be distinguished is turgor pressure which is presumably low in depleted cells but restored when they accumulate K+. Somewhat surprisingly the rate of exchange in the steady state is inhibited by external K+, being over three times higher when K+ is present at 30 pM than at 1mM.49 Control by turgor pressure is demonstrated by examining the effect of reducing turgor in cells maintained in the steady state.49 Turgor is reduced by increasing external osmolarity, a procedure referred to as upshock. In cells expressing Kdp in the steady state, K+ is exchanged at a rate of 35 kmol g-lmin-l representing influx via Kdp and efflux via either Kdp or other paths. (It is not known to what extent Kdp alone mediates K+ exchange in vivo.) Following upshock, efflux remains at control levels of 36 pmol g-'min-I, whereas influx rises within seconds to 74 Fmol g-lmin-l. The mechanism(s) underlying this control are not known, but such control is another possible reason for the presence of three subunits in the KdpABC complex. It could be that the KdpA subunit has specialized not only to bind K+ with high affinity and high specificity, but also to mediate control by turgor. Control of activity by turgor is not unique to Kdp; each of the two other saturable systems, Kup and Trk, show similar behavi0r.4~However, turgor has a smaller effect on Kdp, increasing influx only about 2-fold, whereas in the higher rate Trk system the stimulation is of the order of 5- to 10-fold. Control of Kdp Expression

Kdp activity is not generally seen in cells grown in medium of moderate or elevated K+ concentrations, but is expressed in cells grown at low K+ concentrations and is maximally expressed after growth into K+ limitation. This pattern suggested at first glance that Kdp expression was controlled simply by external K+, but we suspected this explanation was not correct because Kdp was expressed at 5 mM K+ in a trk mutant with reduced K+uptake but not in a wild-type cell. Further analysis was done in a strain with a transcriptional fusion of MpA to the lacZ gene, the fusion abolishing Kdp activity and providing the lacZ product, p-galactosidase, as a convenient indicator of expression of the MpFABC operon.5O In this strain Kdp expression was correlated not with external K+ but with the K+ concentration that became rate-limiting for growth. In a strain wild-type for Trk there was no expression above 3 mM K+, whereas in the mutant lacking both Kup and Trk there was expression below 50 mM K+. Control by internal K+ could also be

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excluded, as this parameter varies with medium osmolarity. At each level of osmolarity and hence of internal K+, one could turn off expression by increasing external K+ so that it was no longer rate limiting for growth. The key experiment implicating turgor was one in which cells were grown at a high K+ concentration where Kdp was not expressed; upon upshock there was transient expression which declined after about 30 minutes when it may be presumed that turgor pressure had been restored. As neither external nor internal K+ concentrations were reduced, the simplest explanation is that turgor pressure itself is sensed to turn on Kdp. It was shown that other osmotic solutes, ionic and neutral, had the same effect whereas the permeant solute glycerol did not.

KdpE

KdDE.-P

ll

I

t C

B

AFP

kdpFAi3C

High (Normal) Turgor

C

B

AFP

kdpFASC

Low Turgor

FIGURE 6. Diagram illustrating the control of expression of the kdpFABC operon by turgor pressure. The KdpD protein is anchored in the cytoplasmic membrane by four transmembrane segments. Changes in stretch in the plane of t h e membrane are presumed to alter the conformation of KdpD, leading to activation of its kinase and phosphotransferase activities when turgor is low. The result is phosphorylation of KdpE which then activates expression of the kdpFABC operon. (Reproduced from ref. 5 with permission.)

A model in which turgor pressure is the parameter sensed to control expression of Kdp fits most of the data, but the evidence is circumstantial rather than direct. Gowrishankarsl has suggested an alternative model in which part of the cytoplasmic pool of K+ is sensed to control Kdp expression. The genetic basis of control resides in the regulatory kdpDE operon adjacent to the kdpFABC operon.'* These operons overlap, as the relatively weak promoter and the transcription start site of kdpDE are within the kdpC gene. Most of the transcripts that begin at the kdpFABC promoter end early in kdpD, but some extend to transcribe the kdpDE operon as well.

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The genes of the regulatory operon encode a pair of proteins5 that are members of a large family of bacterial two-component regulatory systems.6 These systems comprise a larger “sensor” protein that has autokinase activity, phosphotransferase activity, and phosphatase activity, and a smaller “effector” or “response regulator” protein that is phosphorylated by the larger on an aspartate residue, generally can be dephosphorylated by the sensor protein, and in the majority of systems is a positive activator of transcription. There is extensive homology of the soluble KdpE protein to other effector proteins over almost all of its length. Homology of KdpD to other sensors is limited, as is typical; in this group of proteins the conserved residues implicated in kinase activity, including the conserved histidine, are always in the COOH-terminal part of the protein. Interestingly, analysis of seven independent mutants that make Kdp expression partly constitutive shows six are found in the proximal 180 amino residues of KdpD.52This is the region that is unique to KdpD and presumably acts to convert changes in turgor to the signal that turns on expression of the kdpFABC operon. The sequence of the 98.5-kD KdpD protein consists of large soluble COOH- and NHz-terminal regions with four closely spaced membrane-spanning domains in the middleasAs expected from the sequence, Kdp is associated with the inner membrane. This structure is consistent with proteolysis studies indicating that KdpD is resistant to proteases in spheroplasts, but is extensively degraded to small fragments in inside-out vesicles (M. G. Meldorf and W. Epstein, unpublished observations). The scheme of FIGURE 6 indicates the overall topology of KdpD and the phosphorylation that is presumed to mediate its actions. Located in the inner membrane, KdpD could sense stretch in the plane of the membrane, the stretching force reflecting the magnitude of turgor pressure, and such a conformational change could trigger autophosphorylation. Autophosphorylation of KdpD and transfer of the phosphate to KdpE have been demonstrated in v i m (P.Voelkner and K. Altendorf, unpublished observations), supporting the idea that phosphorylation is the way turgor pressure is converted into a signal to alter gene expression. The KdpE protein has been purified and shown to be a DNA binding protein (L. Brandon and W.Epstein, unpublished observations); specific binding to the kdpFABC promoter remains to be demonstrated. Note added in proof: Recent work has provided further information about the promoter and demonstrated many of the features of the regulation of Kdp implied by homologies of the regulatory genes and our preliminary studies. Sugiura et al.53 reported the sequence of the MpFABC promoter region and identified the sites important for regulation by deletion analysis and DNA footprint analysis of KdpE binding in the promoter region. Nakashima et al.54purified KdpE, demonstrated autophosphorylation of KdpD and transfer of phosphate from phospho-KdpD to KdpE. REFERENCES

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Y. A., K. N. DZHANDZUGAZYAN, S. V. LUTSENKO, A. A. MUSTAYEV & N. N. 24. OVCHINNIKOV, MODYANOV.1987. Affinity modification of El form of Na+,K+-ATPase revealed Asp-710 in the catalytic site. FEBS Lett. 217: 111-116. & K. &TENDOW. 1992. The phosphorylation site of the Kdp25. PUPPE,W., A. SIEBERS ATPase of Escherichia coli. Site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit. Mol. Microbiol. In press. M., A. SCHLESSER & A. GOFFEAU.1987. Mutation of a conserved glycine 26. GHISLAIN, residue modifies the vanadate sensitivity of the plasma membrane H+-ATPase from Schizosaccharomycespombe.J. Biol. Chem. 262 17549- 17555. J. P., B. VILSEN,E. LEBERET& D. H. MACLENNAN. 1989. Functional 27. ANDERSEN, consequences of mutations in the p-strand sector of the Ca2+-ATPaseof sarcoplasmic reticulum. J. Biol. Chem. 264: 21018-21023. & M. YOSHIDA. 1986. The active site structure of the Na+/K+28. OHTA,T., K. NAGANO transporting ATPases: Location of the 5'-@-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin. Proc. Natl. Acad. Sci. USA 83: 2071-2075. A. 1988. Kalium-Transport bei Escherichia coli: Funktionelle, immunologische 29. SIEBERS, und topographische Untersuchungen der Kdp ATPase. Doctoral thesis, University of Osnabriick. P. J. F. & M. C. J. MAIDEN.1990. Homologous sugar transport proteins in 30. HENDERSON, Escherichia coli and their relatives in both prokaryotes and eukaryotes. Phil. Trans. R. SOC.Lond. B. 326 391-410. H. E., E. RIBI & P. D. ROEPE.1990. p-galactoside transport in E. coli: A 31. KABACK, functional dissection of the lac permease. Trends Biochem. Sci. 15: 309-314. 32. VON HEIJNE,G. 1986. The distribution of positively charged residues in bacterial inner membrane proteins correlates with the transmembrane topology. EMBO J. 5: 30213027. G. E., L. K. LANE & J. B. LINGREL. 1986. Amino acid sequence of the p-subunit of 33. SHULL, the (Na+ + K+ )ATPase deduced from a cDNA. Nature 312 429-431. G. E. 1990. cDNA cloning of the p-subunit of the rat gastric H,K-ATPase. J. Biol. 34. SHULL, Chem. 265 12123-12126. & R. A. FARLEY. 1990. The sodium pump needs its p A. A., K. GEERING 35. MCDONOUGH, subunit. FASEB J. 4: 1598-1605. 36. SIEBERS, A. & K. ALTENDORF. 1989. Characterization of the phosphorylated intermediate of the K+-translocatingKdp ATPase from Escherichia coli. J. Biol. Chem. 264: 58315838. J. W. 1986. The identification and analysis of the regulatory genes of the Kdp 37. POLAREK, system of Escherichia coli. Doctoral thesis, University of Chicago. L. A. 1981. Organization of the Kdp potassium transport system of Escherichia 38. LAIMINS, coli. Doctoral thesis, University of Chicago. M. D., C. G. MILLER & M. E. MAGUIRE.1991. The mgfBMg2+transport locus of 39. SNAVELY, Salmonella typhimurium encodes a P-type ATPase. J. Biol. Chem. 266: 815-823. 40. SIEBERS, & K. ALTENDORF. 1988. K+-ATPase from Escherichia coli: A., L. WIECZOREK Isolation and characterization. Methods Enzymol. 157: 668-680. 41. SIEBERS, A., R. KOLLMAN, G. DIRKES& K. ALTENDORF. 1992. Rapid, high-yield purification and characterizationof the K+ translocating Kdp-ATPase from Escherichia coli. J. Biol. Chem. 267: 12717-12721. I. M. & S. J. D. KARLISH.1975.The sodium pump. Annu. Rev. Physiol. 37: 13-55. 42. GLYNN, 43. NAPRSTEK, J., M. 0. WALDERHAUG & W. EPSTEIN.1992. Ann. N.Y. Acad. Sci. This volume. 44. EPSTEIN, & J. HESSE.1978. A K+-transport ATPase in Escherichia coli. W., V. WHITELAW J. Biol. Chem. 253 6666-6668. 45. JAIMINS, L. A. 1981. Organization of the Kdp potassium transport system of Escherichia coli. Doctoral thesis, University of Chicago. 46. HUGENHOLTZ, J., J. HONG& H. R. KABACK.1981. ATP-driven active transport in right-side-out membrane vesicles. Proc. Natl. Acad. Sci. USA 7 8 3446-3449. W. 1986. Osmoregulation by potassium transport in Escherichia coli. FEMS 47. EPSTEIN, Microbiol. Rev. 3 9 73-78. D. B., F. B. WATERS& W. EPSTEIN.1976. Cation transport in Escherichia coli. 48. RHOADS, VIII. Potassium transport mutants. J. Gen. Physiol. 67: 325-341.

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DISCUSSION

JACKKAPLAN(University of Pennsylvania, Philadelphia, PA): D o you have an estimate of the number of K+ ions transported per ATP molecule hydrolyzed? WOLFGANGEPSTEIN: Kdp has not yet been successfully reconstituted into vesicles, so our only insight is based on energetics. We find that Kdp can create a K+ concentration of - 4 x 106:l; this would allow at most 2 K+ to be transported per ATP, assuming stoichiometry does not change with the load. SERGIOPAPA (University of Bari, Bari, Italy): You briefly mentioned that Kdp ATPase might work in a dimeric state. Could you specify the evidence supporting this possibility? In fact, a dimeric mode of operation would have important implications on the way in which transmembrane segments involved in transport are organized. WOLFGANGEPSTEIN:We found in 1970 that two mutations that affect the KdpA subunit complement each other. Such intracistronic complementation is considered reliable evidence that the functional state of the product is oligomeric. We postulate a dimeric state as the simplest oligomer. In our model, we assume that each monomer forms a K+ channel, rather than that the K+ channel is found between the two monomers. DON MENICK(Medical University of South Carolina, Charleston, SC): Are the phosphoenzyme formation experiments done in membrane vesicles? Have you looked at phosphorylation of Kdp by inorganic phosphate? We have characterized a prokaryotic Ca2+ATPase which is different from the classical P-type reaction cycle in that it can form a phosphoenzyme from inorganic phosphate in the presence of Ca*+. W e are curious if other prokaryotic P-type ATPases have differences in, or alternative reaction cycles from, the eukaryotic P-type ATPases. W e can form the phosphorylated intermediate in crude WOLFGANGEPSTEIN: membranes or with purified, detergent-solubilized enzyme. W e have not yet examined formation of the intermediate from inorganic phosphate. (Institute of Physico-Chemical Biology, Moscow, Russia): Are some V. SKULACHEV other ions transported together with K+ or against it by Kdp, or is this the K+ uniport? WOLFGANGEPSTEIN:We d o not know if Kdp transports another ion besides K+. There is no Na+ requirement for Kdp transport in vivo or for Kdp ATPase activity in vitro. Kdp may mediate proton export, but preliminary experiments in vesicles have not provided any evidence for it.