©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Membrane Topology Analysis of the Sensor Kinase KdpD of Escherichia coli(*)

(Received for publication, May 30, 1995; and in revised form, August 21, 1995)

Petra Zimmann (§) Wolfram Puppe Karlheinz Altendorf (¶)

From the Universität Osnabrück, Fachbereich Biologie/Chemie, D-49069 Osnabrück, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The expression of the kdpFABC operon, coding for the K-translocating Kdp-ATPase, is under the control of the two regulatory proteins KdpD and KdpE, which belong to the group of sensor kinase/response regulator systems. The topology of the KdpD protein in the cytoplasmic membrane was investigated using LacZ and PhoA fusions at different sites within the polypeptide chain and by treating spheroplasts in the presence or absence of Triton X-100 with the protease kallikrein. The results revealed that KdpD has four membrane-spanning segments in the middle of the polypeptide chain, whereas N and C terminus are both cytoplasmic.


INTRODUCTION

K ions play a crucial role in maintaining turgor in bacterial cells (Epstein, 1986). Therefore, bacteria have established several types of K influx and efflux systems as well as secondary porters and stretch-activated channels to adjust the intracellular K concentration in response to changes in the osmolarity of the medium (Bakker, 1993). In the case of Escherichia coli the Kdp-ATPase, an inducible, high affinity K uptake system (K for transport: 2 µM; Epstein et al., 1978) is expressed as an emergency system under growth conditions, where the three constitutive K uptake systems TrkH, TrkG, and Kup (Bossemeyer et al., 1989; Dosch et al., 1991) are unable to maintain the required cellular pool of K. The Kdp-ATPase is composed of the three membrane-bound subunits KdpA, KdpB, and KdpC (Laimins et al., 1978). The structural genes are organized in the kdpFABC operon (Hesse et al., 1984), whereby the function of the open reading frame kdpF is still unknown (Altendorf et al., 1992). The adjacent kdpDE operon codes for two proteins that regulate the expression of the kdpFABC operon (Polarek et al., 1992). KdpD is a membrane-bound protein (98.7 kDa), whereas KdpE is a cytoplasmic protein (25.2 kDa) (Walderhaug et al., 1992). Both proteins belong to the class of sensor kinase/response regulator systems, which are characterized by a homologous signal-transduction pathway for the adaptive response of environmental stimuli (Parkinson and Kofoid, 1992).

In all cases examined to date, the signal transduction of the sensor kinase/response regulator systems occurs via a phosphorylation cascade. The phosphorylation of the KdpD protein in vitro, most probably at the conserved residue His-673, and the subsequent transfer of the phosphoryl group to the response regulator KdpE was shown recently (Nakashima et al., 1992; Voelkner et al., 1993). Furthermore, the interaction of KdpEP with the kdpFABC promoter, resulting in a 10-fold increase of the transcription of the kdpFABC operon, was demonstrated (Nakashima et al., 1993). For KdpD the stimulus seems to be a decrease in turgor (Laimins et al., 1981; Epstein, 1992). However, further observations lend support to the notion that turgor is apparently not the sole regulatory signal for the expression of kdp (Csonka and Hanson, 1991; Asha and Gowrishankar, 1993).

Compared to other sensor kinases, the transmitter domain (C-terminal part) of KdpD is highly conserved, whereas the input domain shows no homology to any other protein sequenced so far. In the case of KdpD the input domain, which in general is responsible for sensing the signal, is rather large, comprising the N-terminal region of 400 amino acids, the membrane-spanning segments, and approximately 200 amino acids of the C-terminal region. In order to understand the mechanism of signal perception, a topological analysis of KdpD in the membrane has been performed using protein fusions to PhoA and LacZ and protease susceptibility experiments.


EXPERIMENTAL PROCEDURES

Materials

All chemicals used were of analytical grade. Nitrocellulose membranes (0.45 µm) were obtained from Schleicher & Schüll (Dassel, Germany (FRG)). T4 DNA-ligase, alkaline phosphatase, Klenow fragment of DNA polymerase I, T4 polynucleotide kinase, restriction endonucleases, and dNTPs were purchased from Boehringer (Mannheim, FRG), Life Technologies, Inc. (Eggenstein, FRG), Biolabs (Schwalbach, FRG), or Pharmacia (Freiburg, FRG). Agarose was obtained from Roth (Karlsruhe, FRG). Goat anti-rabbit IgG alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Biomol (Hamburg, FRG), isopropyl-beta-D-thiogalactoside from Serva (Heidelberg, FRG), and leupeptin, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, o-nitrophenyl-beta-D-galactoside, and p-nitrophenyl phosphate were obtained from Sigma (Munich, FRG). Tfl thermostable DNA polymerase was purchased from BIOzym (Hameln, FRG), while the T7 sequencing kit was obtained from Pharmacia. The protease kallikrein and anti-beta-galactosidase antibody were obtained from Boehringer (Mannheim, FRG). Sheep-anti-(mouse Ig) horseradish peroxidase-linked whole antibody and the ECL-chemiluminescence Western blotting detection reagents were purchased from Amersham Buchler (Braunschweig, FRG). Oligonucleotides for sequencing and PCR^1 were synthesized by Dr. H. Lill (University of Osnabrück).

Bacterial Strains, Plasmids, and Media

E. coli strains and plasmids used are listed in Table 1. All strains are derivatives of E. coli K12. Strain TKV2209 carries a deletion from EcoRV (bp 4699; see Fig. 1) in kdpD to BamHI (bp 7614) in kdpE. The deletion was constructed in vitro on a plasmid, and the transfer to the chromosome was performed using the polA technique as described by Gutterson and Koshland(1983). KML complex medium was prepared as described (Epstein and Davies, 1970), and antibiotics were used in the following concentrations: 25 µg/ml chloramphenicol, 50 µg/ml ampicillin, and 100 µg/ml carbenicillin.




Figure 1: Construction of kdpD deletions. Part of the kdpD gene, which was used to construct the desired plasmids, is shown in more detail. The position of the primers D3, D4, D5, D6, and D7 within the kdpD sequence are indicated in the upper part. Black arrows represent identical (D3) or complementary (D4, D5, D6, D7) sequences to kdpD, whereas the dotted part of the arrow represents newly introduced sequences comprising BamHI (B) and EcoRV (EV) recognition sites. The orientation of the arrows indicates the direction of polymerization during the PCR. The numbered open bars (1-4) represent the sequence of the predicted membrane-spanning helices (Walderhaug et al., 1992). Numbered arrows at the bottom of the pPV2 sequence indicate the positions where the complementary sequences within the primers D4-D7 stops. The positions (in bp) of EcoRV sites used for the construction are displayed (EV 4699 and EV 5908). The numbering of the nucleotides is given in bp according to the kdpDE sequence published by Walderhaug et al.(1992). The four sequences in the lower part of the figure show the constructed deletion plasmids pPV2-D4 to pPV2-D7. The hatched lines indicate the fusion of the newly introduced EcoRV site to the EcoRV site at bp 5908 (for the cloning strategy, see ``Experimental Procedures''). BamHI sites used for the construction of the lacZ and phoA fusions are indicated by arrows designated B. The truncated KdpD proteins and the amino acids lacking therein are given on the right.



Recombinant DNA Techniques and PCR

For preparation and handling of recombinant DNA and for transformation of E. coli cells, standard procedures were used (Sambrook et al., 1989; Ausubel et al., 1987). Treatment of DNA with enzymes (restriction enzymes, alkaline phosphatase, Klenow fragment of DNA polymerase I, polynucleotide kinase, ligase) was carried out under conditions recommended by the suppliers. Prior to ligations vector DNA fragments were treated with alkaline phosphatase. DNA fragments were recovered from agarose gels using the Gene-clean kit from Dianova (Hamburg, FRG). Sequencing of double-stranded DNA was performed by the dideoxynucleotide chain termination method (Sanger et al., 1977) using a Pharmacia T7 sequencing kit. Polymerase chain reactions were carried out with the Tfl thermostable DNA polymerase, and PCR products were purified using the Magic-Prep kit from Promega (Madison, WI).

Construction of Different kdpD Deletions

Four kdpD deletions were generated by using the PCR method followed by two additional subcloning steps. Primers used for PCR and their positions within the kdpD gene are shown in Fig. 1. Primer D3 (5`-CCCGTCATCCCAAACGCTG-3`) corresponds to bp 4673-4692 just upstream of the first EcoRV site in the kdpD gene. The sequence of primer D4 (3`-GTTCACCGCACATGTCCTAGGGCTATAG-5`) is in the 3` part complementary to bp 5497-5511. In the 5` part (underlined), a new sequence containing a BamHI and an EcoRV restriction site was created. These new restriction sites were used to construct ``in frame'' KdpD deletion (EcoRV) or fusion (BamHI) proteins. The sequences of primer D5 (3`-CTACCGCAAACTACGCCTAGGGCTATAG-5`), primer D6 (3`-CGATAAAATACCTGCCCTAGGGCTATAG-5`), and primer D7 (3`-GCGGGGTGCGCCGTGCCTAGGGCTATAG-5`) are in the 3` part complementary to the sequences of bp 5578-5592 (D5), bp 5638-5652 (D6), and bp 5710-5724 (D7), respectively. The 5` parts of the primer D5, D6, and D7 (underlined) carry the same new BamHI-EcoRV recognition sequence as primer D4. PCR amplification was performed using plasmid pPV2 as template with the primer pairs D3 and D4, D3 and D5, D3 and D6, as well as D3 and D7. The resulting PCR products were purified and treated with Klenow fragment and T4 polynucleotide kinase. For more convenient handling, the PCR products were first cloned in the SmaI site of vector pUC19. The resulting plasmids were named pUCD4, pUCD5, pUCD6, and pUCD7. In a second step the four different kdpD deletion plasmids were constructed. For this purpose the kdpD` containing the 1209-bp EcoRV fragment of plasmid pPV2 was replaced by the EcoRV-digested PCR fragments of plasmids pUCD4, pUCD5, pUCD6, and pUCD7, respectively, resulting in plasmids pPV2-D4, pPV2-D5, pPV2-D6, and pPV2-D7 (see Fig. 1). Verification of the four kdpD deletions was achieved by restriction analysis and plasmid sequencing.

Construction of kdpD-lacZ and kdpD-phoA Fusions

The fusions of the kdpD gene to the lacZ or the phoA gene were generated at five different sites within the kdpD gene. For this purpose the 3068-bp BamHI fragment of plasmid pZ19 (Haardt, 1993) containing the lacZ reporter gene was used, which is deleted for the first 19 nucleotides of the gene, but includes the stop codon. The phoA reporter gene was taken from plasmid pSWFII (Ehrmann et al., 1990). The 1300-bp BamHI fragment of pSWFII is deleted for the first 78 nucleotides of phoA coding for the signal sequence and lacks a stop codon at the 3` end.

The first fusion point was the EcoRV site at bp 4699 at the beginning of the kdpD gene (Fig. 1). Plasmid pPV14 (kdp`D-lacZ) and pPV15 (kdp`D-phoA-kdpD`) were constructed by replacing the 1209-bp EcoRV fragment (bp 4699-5908, see Fig. 1) with the lacZ and phoA fragment, respectively. Prior to that the BamHI ends of the fragment DNA had been filled in using Klenow fragment. For the other fusion points, the newly introduced BamHI site in the deleted kdpD genes of plasmids pPV2-D4, pPV2-D5, pPV2-D6, and pPV2-D7 were used (Fig. 1). For that purpose the BamHI-restricted lacZ and phoA fragment, respectively, were ligated with the BamHI vector part of plasmid pPV2-D4 to pPV2-D7. The resulting plasmids were named pPV2-D4L and pPV2-D4P to pPV2-D7L and pPV2-D7P, respectively. In this cloning step the kdpD gene downstream of the BamHI site and most of the kdpE gene in plasmids pPV2-D4 to pPV2-D7 were deleted due to a second BamHI site (bp 7614) at the end of the kdpE gene.

In the case of lacZ fusions, strain TKV2209 (lacZ) was transformed with the ligation mixture and recombinant plasmids were selected on KML agar plates containing chloramphenicol and the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (40 mg/liter). In contrast, strain DHB4 (phoA) was used for the phoA fusion plasmids and transformants were selected on KML agar plates with chloramphenicol, isopropyl-beta-D-thiogalactoside (1 mM), and the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (40 mg/liter). The constructed plasmids were tested by restriction analysis and by immunodetection of the synthesized fusion proteins with antibodies raised against KdpD, LacZ, and PhoA.

Assay of beta-Galactosidase and Alkaline Phosphatase Activity

For the beta-galactosidase assay, strain TKV2209 harboring plasmids pPV2, pPV14, and pPV2-D4L to pPV2-D7L, respectively, was grown in KML complex medium with chloramphenicol to logarithmic phase and the beta-galactosidase activity was measured as the rate of o-nitrophenyl-beta-D-galactoside hydrolysis in permeabilized cells according to Miller(1972). Therefore, 1 ml of cells were washed in 50 mM phosphate buffer, pH 7.2. After addition of 20 µl toluol, the suspension was incubated at 37 °C for 5 min. The reaction was started by addition of 50 µl of o-nitrophenyl-beta-D-galactoside (20 mM), and after development of sufficient yellow color the reaction was stopped with 1.5 ml of Na(2)CO(3) (200 mM). Before measuring the absorbance at 420 nm, cell debris were removed by centrifugation for 10 min at 13,000 times g in a microcentrifuge.

The determination of AP activity was performed with cells of strain DHB4 harboring plasmids pPV2, pPV15, and pPV2-D4P to pPV2-D7P, respectively, grown to logarithmic phase in KML complex medium with chloramphenicol and 1 mM isopropyl-beta-D-thiogalactoside. The rate of p-nitrophenyl phosphate hydrolysis in permeabilized cells was measured according to Michaelis et al.(1983). In this case 0.5 ml of cells were diluted with 0.5 ml of buffer A (10 mM Tris/HCl, pH 8.0, 150 mM NaCl) and washed twice with the same buffer. Subsequently, 0.1 ml of the sample were added to 0.9 ml of buffer B (1 mM Tris/HCl, pH 8.0, 1 mM ZnCl(2)). Cells were permeabilized by the addition of 25 µl of SDS (0.1%) and 25 µl of chloroform and incubated at room temperature for 5 min. The enzymatic reaction was started by the addition of 0.1 ml of p-nitrophenyl phosphate (0.4% in 1 M Tris/HCl, pH 8.0) and incubated at 37 °C, until sufficient yellow color was observed. The reaction was stopped by the addition of 120 µl of 2.5 M K(2)HPO(4), and cell debris were removed by centrifugation for 10 min at 13,000 times g before measuring the absorbance at 420 nm. In both cases units of enzyme activity are defined as OD times 1000/t times v times OD (Miller, 1972; Brickmann and Beckwith, 1975), where OD represents the reaction mixture, OD the cell density just before the assay, t the reaction time in minutes, and v the volume of cell culture used for the assay in ml.

Preparation of Everted Membrane Vesicles

Cells were grown in KML complex medium supplemented with chloramphenicol to an optical density (OD) of approximately 1.0. Membranes were prepared according to Siebers and Altendorf(1988), but instead of using a Ribi Press fractionator cells were disrupted by sonification for 3 min at 4 °C on ice (duty cycle, 50%; Branson sonifier). Furthermore, the washing step in low ionic strength buffer was omitted and buffers were changed from Hepes/Tris to Tris/HCl.

Preparation of Spheroplasts

Exponentially grown cells (OD = 0.6) were washed once in 33 mM Tris/HCl, pH 8.0, 100 mM KCl, and 5 mM MgSO(4). Subsequently, cells were resuspended in the same buffer plus 40% (w/v) sucrose. Spheroplasts were prepared by the addition of EDTA (20 mM final concentration) and lysozyme (100 µg/ml) and incubated at 4 °C for 15 min. The spheroplasts were used immediately without further washing.

Proteolysis with Kallikrein

Spheroplasts alone or in the presence of 0.5% Triton X-100 were used in the corresponding buffer at a protein concentration of 0.5 mg/ml. Proteolysis was carried out with kallikrein (100 µg/ml) for 1 h at 37 °C. The reaction was stopped by the protease inhibitor leupeptin (1 µM), followed by the precipitation of the protein with 10% trichloroacetic acid. The precipitated proteins were washed once with acetone (4 °C), dried under a stream of nitrogen, and further used for SDS-PAGE.

Analytical Procedures

Protein concentrations were determined according to Hartree(1972). SDS-PAGE was performed as described by Lugtenberg et al.(1975) using 7,5% or 9% polyacrylamide gels. The electrophoretic transfer of proteins from SDS-polyacrylamide gels to nitrocellulose membranes and immunodetection with anti-KdpD antiserum (dilution 1:100,000, Voelkner et al., 1993) and alkaline phosphatase-conjugated secondary antibody was carried out as described by Voelkner et al.(1993), except that the blocking buffer was changed from 3% bovine serum albumin to 5% (w/v) powdered skim milk. Furthermore, in the case of anti-LacZ as primary antibody (Boehringer), the peroxidase-conjugated secondary antibody was detected with the detection reagents of the ECL-Western blotting system as recommended by the supplier.


RESULTS

It has already been shown that the KdpD protein is located in the cytoplasmic membrane of E. coli (Walderhaug et al., 1992; Voelkner et al., 1993). The hydrophobicity plot according to the algorithm of Kyte and Doolittle(1982) revealed that KdpD is predominantly of hydrophilic character except for four narrow-spaced, membrane-spanning segments in the middle of the primary structure (Fig. 2). However, experimental evidence in support of this topological model was still lacking.


Figure 2: Hydropathy profile of the KdpD protein. The average local hydrophobicity at each residue according to the algorithm of Kyte and Doolittle(1982), using a window of 15 amino acids, is plotted on the vertical axis versus the residue number on the horizontal axis.



Construction of kdpD Deletion Plasmids

In order to fuse PhoA or LacZ to the proposed periplasmic or cytoplasmic loops of KdpD, various deletions comprising the central membrane-spanning helices were generated, together with the introduction of a convenient BamHI site as described under ``Experimental Procedures.'' The PCR products used for the cloning steps and the resulting plasmids carrying the deleted kdpD genes are displayed in Fig. 1. Plasmid pPV2-D4 carries the 822-bp EcoRV fragment of the PCR product obtained with primer pair D3 and D4 instead of the original 1209-bp EcoRV fragment of pPV2. In analogy the original EcoRV fragment was replaced by a 903-bp EcoRV fragment (primers D3 and D5) in pPV2-D5, by a 963-bp EcoRV fragment (Primers D3 and D6) in pPV2-D6, and by a 1035-bp EcoRV fragment (primers D3 and D7) in pPV2-D7, respectively. DNA sequencing of the deletions in plasmids pPV2-D4 to pPV2-D7 was performed to verify the correct positioning of the newly created BamHI sites and to proof that all constructions are ``in frame'' deletions.

Detection of the Truncated KdpD Proteins

Everted membrane vesicles of strain TKV2209 harboring plasmid pPV2-D4, pPV2-D5, pPV2-D6, and pPV2-D7, respectively, were prepared as described under ``Experimental Procedures.'' The synthesis of the four proteins KdpD4, KdpD5, KdpD6, and KdpD7 was confirmed by immunoblotting using polyclonal antibodies raised against wild type KdpD protein (Fig. 3). Their predicted sizes, ranging from 85 kDa to 93 kDa, are in good agreement with those calculated from the immunoblot. The results also demonstrated that all proteins carry ``in frame'' deletions. Furthermore, the truncated proteins could all be detected in the membrane fraction. As already shown by Puppe et al.,^2 KdpD proteins lacking all four membrane-spanning helices, like the KdpD4 protein, are still associated with, but not integrated into the membrane, probably due to an interaction of a stretch of hydrophobic amino acids in the N-terminal part of KdpD with the membrane. However, the amount of the four truncated proteins synthesized is different (Fig. 3), although their genes are expressed from the same plasmid background. The protein level is the highest for KdpD4, followed by KdpD6, and decreased from KdpD7 to KdpD5. This phenomenon could possibly be explained by the different stability of the truncated forms in the cytoplasmic membrane.


Figure 3: Detection of truncated Kdp proteins. Everted membrane vesicles from strain TKV2209 carrying plasmid pPV2-D4, pPV2-D5, pPV2-D6, and pPV2-D7, respectively, were prepared as described under ``Experimental Procedures.'' Proteins (30 µg/lane) were separated on 7.5% SDS-PAGE, and a polyclonal anti-KdpD antiserum was used for immunoblotting (dilution 1:100,000). The protein standard and a KdpD reference were applied to the first lane.



Construction of KdpD-LacZ and KdpD-PhoA Fusion Proteins

To investigate the membrane topology of the KdpD protein in more detail, gene fusions of kdpD ``in frame'' to lacZ and phoA were constructed as described under ``Experimental Procedures.'' The five different fusion points are shown in Table 2and Fig. 1. For the first fusion at residue Asp-127, the EcoRV site at bp 4699 was used. The resulting fusion protein Kdp`D-LacZ contains only the kdpD sequence upstream of the EcoRV site, whereas the fusion protein Kdp`D-PhoA-KdpD` also contains the kdpD sequence proximal to the second EcoRV site (residue Ile-531 to Met-894), because the stop codon of the phoA gene is lacking. The 130-kDa Kdp`D-LacZ protein could be detected with the anti-LacZ antiserum (Fig. 4). However, the fusion protein seems to be unstable, because the second detectable, faster moving band has the mobility of the beta-galactosidase. The 105-kDa Kdp`D-PhoA-KdpD` protein could be detected with the anti-KdpD antiserum, since the antigenic C-terminal region of KdpD is still present (data not shown). The four other fusions at residues Gln-398, Ala-425, Arg-445, and Thr-469, respectively, were constructed by using the newly created BamHI site in the deleted kdpD genes of plasmids pPV2-D4, pPV2-D5, pPV2-D6, and pPV2-D7. As shown in Fig. 1and Fig. 7, these fusion points are lying immediately before or between the predicted membrane-spanning helices. The corresponding fusion proteins Kdp`D4-LacZ, Kdp`D5-LacZ, Kdp`D6-LacZ, and Kdp`D7-LacZ, with predicted sizes between 155 and 165 kDa, could all be detected with the anti-LacZ antiserum (Fig. 4). It can clearly be seen that the level of the Kdp`D4-LacZ protein is elevated in comparison to the other fusion proteins.




Figure 4: Detection of KdpD-LacZ fusion proteins. Exponentially growing cells of strain TKV2209 harboring plasmids pPV2 (KdpD), pPV14 (Kdp`D-LacZ), and pPV2-D4L (Kdp`D4-LacZ) to pPV2-D7L (Kdp`D7-LacZ), respectively, were harvested by centrifugation and resuspended in gel electrophoresis sample buffer. ``Whole cell'' proteins (40 µg/lane) were separated on 7.5% SDS-PAGE, and a monoclonal anti-LacZ antiserum (Boehringer) was used for immunoblotting.




Figure 7: Topology of the KdpD protein. The illustrated model was derived from both hydropathy analysis and studies with LacZ/PhoA fusion proteins as described in the text. Transmembrane helices are displayed as white boxes, and the first and last residue of the predicted hydrophobic region are indicated. The positions of the LacZ/PhoA fusion point are marked by arrows, and the corresponding amino acid residue is indicated. The net charge of the hydrophilic regions before, between or after the hydrophobic domains, taking up to 10 amino acids into account, is circled. Arg, Lys, and His are considered positive, Glu and Asp are considered negative.



The detection of the PhoA fusion proteins, Kdp`D4-PhoA to Kdp`D7-PhoA, with predicted sizes ranging from 95 to 105 kDa, turned out to be more difficult. Only the Kdp`D5-PhoA fusion protein could clearly be detected with an anti-PhoA antiserum (data not shown). This phenomenon could be explained by the instability of these fusion proteins or by the inability of the anti-PhoA antibodies as well as the anti-KdpD antibodies to detect their antigen within the fusion protein.

Enzyme Activities of the Fusion Proteins

In order to determine the location of the fusion points, leading to the overall topology of the KdpD protein, the AP and beta-galactosidase activities were measured as described under ``Experimental Procedures''; the results are shown in Table 2. In this context, it should be noted that the activities are normalized by the amount of permeabilized cells and not by the amount of fusion protein.

It has already been well established that the alkaline phosphatase is inactive in the cytoplasm (Manoil et al., 1990), whereas the beta-galactosidase remains inactive in the periplasm (Lee et al., 1989). The fusion proteins Kdp`D-LacZ/Kdp`D-PhoA-KdpD`, Kdp`D4-LacZ/Kdp`D4-PhoA, and Kdp`D6-LacZ/Kdp`D6-PhoA exhibited high levels of beta-galactosidase, but very low AP activity. For that reason we concluded that the fusion residues Asp-127, Gln-398, and Arg-445 are located in cytoplasmic regions of the KdpD protein. In contrast, the fusion proteins Kdp`D5-LacZ/Kdp`D5-PhoA showed high AP activity, but low beta-galactosidase activity, consistent with a periplasmic exposure of the fusion residue Ala-425. The only fusion without a definite result is that at position Thr-469. The activities of both fusion proteins Kdp`D7-LacZ/Kdp`D7-PhoA are only slightly elevated compared to the basal level.

Judging from the immunoblot (Fig. 4), the concentration of the KdpD-LacZ fusion proteins in the cell lysates are almost identical for Kdp`D5-LacZ to Kdp`D7-LacZ. In the case of Kdp`D4-LacZ, the protein concentration is 3-4 times higher, whereas in the case of Kdp`D-LacZ the protein concentration is roughly 2 times lower compared to the other three fusion proteins. Taking these different protein levels into account, the beta-galactosidase activity of Kdp`D-LacZ would be higher, that of Kdp`D4-LacZ would be 3-4 times lower and that of the other three would not change at all. Based on this kind of normalization of enzyme activity, the same conclusions can still be drawn for the topology of KdpD as was the case for the normalization based on the amount of permeabilized cells (Table 2).

Protease Treatment of KdpD

Further evidence that the N-terminal part of KdpD is located in the cytoplasm stems from protease susceptibility studies. In both the N- and the C-terminal part of KdpD, but not in the central hydrophobic region, seven potential cleavage sites (before Phe-Arg or Leu-Arg) for kallikrein can be found. Treatment of spheroplasts permeabilized by Triton X-100 with kallikrein exhibits an complete degradation of KdpD, whereas in the case of spheroplasts without Triton X-100 the sensor kinase is not touched at all (Fig. 5). In contrast, trypsin causes degradation of KdpD even in spheroplasts (data not shown), since potential cleavage sites for this protease reside in the postulated periplasmic loops of the central hydrophobic region.


Figure 5: Protease treatment of KdpD. KdpD-containing spheroplasts and spheroplasts permeabilized with Triton X-100 from strain TKR2000/pPV5 were prepared as described under ``Experimental Procedures.'' Samples (0.5 mg/ml) with (+) or without(-) the protease kallikrein (100 µg/ml) were incubated for 1 h at 37 °C. Proteins (20 µg/lane) were separated on 9% SDS-PAGE and a polyclonal anti-KdpD antiserum was used for immunoblotting.




DISCUSSION

The sensor kinase KdpD is related to a group of sensor kinases, like PhoR (Makino et al., 1986) and EnvZ (Comeau et al., 1985), which exhibit a modular design of an N-terminal, mostly periplasmic input domain located between two membrane-spanning segments. Sequence homology of KdpD to other sensor kinases begins at approximately residue 660 with the C-terminal transmitter domain. This domain carries the five characteristic sequence motifs (blocks H, N, G1, F, and G2; Parkinson and Kofoid(1992)), and possesses autokinase and phosphotransferase activity (Nakashima et al., 1992; Voelkner et al., 1993; Altendorf et al., 1994). Therefore, the C-terminal domain of KdpD, which must interact with ATP and the cytoplasmic protein KdpE, is surely located in the cytoplasm. That also applies to the short hydrophobic region from residue 840 through 852, which appears as a prominent peak in the hydrophobicity plot (Fig. 2).

KdpD possesses an unusually extended N-terminal input domain, which is mostly hydrophilic except for the central membrane-spanning helices (Fig. 2). This region does not show homology to any other protein sequenced so far. Our working model (Fig. 6A) suggests that this domain of about 400 amino acid residues is also cytoplasmic and that the central hydrophobic region consists of four membrane-spanning segments (Walderhaug et al., 1992). However, the N-terminal region possesses a stretch of 17 hydrophobic amino acids from residues Val-27 to Ala-43, which corresponds to the first peak in the hydrophobicity plot (Fig. 2). As predicted by the algorithm of Rao and Argos(1986), this region could also be a membrane-spanning helix. Therefore, a second membrane model of KdpD, in which the N-terminal input domain is mostly periplasmic is possible (Fig. 6B). As a consequence of the cytoplasmic localization of the C terminus, in this model only three membrane-spanning segments could exist in the middle of the protein, as predicted by the algorithm of Rao and Argos (1986; data not shown).


Figure 6: Models of KdpD. Two possible models (A and B) for the topology of the KdpD protein in the cytoplasmic membrane are shown. The postulated phosphorylation site His-673 is indicated.



To be able to distinguish between these two models for KdpD, PhoA and LacZ fusion proteins have been used, which are only active in the periplasm or in the cytoplasm (for an overview, see Traxler et al.(1993)). The five fusion points were chosen in such a way that the number of the central membrane-spanning helices, and in particular, the location of the N-terminal domain could be clearly established. We provide experimental evidence for a cytoplasmic localization of the N-terminal domain of KdpD leading to the topology model of KdpD as given in Fig. 7.

In detail, the high beta-galactosidase and low AP activities of the KdpD-LacZ and KdpD-PhoA fusions at residues Asp-127, Gln-398, or Arg-445 suggest a cytoplasmic location of these fusion points. On the other hand, the low beta-galactosidase activity of the Kdp`D5-LacZ fusion at residue Ala-425 or of Kdp`D7-LacZ at residue Thr-469 indicates a location of the fusion points at the periplasm. In case of the PhoA fusions, high AP activity could clearly be demonstrated for the Kdp`D5-PhoA fusion, indicating again a periplasmic location of that fusion point. However, Kdp`D7-PhoA exhibited low instead of high AP activity. This latter observation can possibly be explained by the inability of the Kdp`D7 fusions to export the reporter proteins due to ineffective or deleted topogenic signals or due to premature splitting of the reporter proteins. These arguments also apply to the slightly elevated beta-galactosidase activity of Kdp`D7-LacZ. These kind of problems are already known from topological studies of other membrane proteins. In the case of the LacY protein (Calamia and Manoil, 1990), an alkaline phosphatase fusion to a known periplasmic domain has low AP activity presumably because it follows a poor export signal. Detailed studies revealed that a charged residue in the membrane-spanning segment N-terminal to this periplasmic domain limit export of the reporter protein (Calamia and Manoil, 1992). In the case of KdpD only the third membrane-spanning segment contains a charged residue. Therefore, in analogy, the low AP activity of Kdp`D7-PhoA may also be explained by an inefficient export of PhoA fused to the periplasmic domain C-terminal to the third membrane-spanning segment, since the interaction of the third membrane-spanning segment with the fourth transmembrane helix of KdpD might be necessary for a correct membrane insertion. When sequences located C-terminal of the fusion point are required for a proper topological assembly, as demonstrated for the MalK protein (McGovern et al., 1991), sandwich gene fusions (Ehrmann et al., 1990) are sometimes a better tool to determine the topology of a given protein than fusions in which the C-terminal part of the protein is replaced by the reporter protein. Furthermore, problematic fusions could be counteracted by shifting the fusion point by some amino acids as demonstrated for the LacY protein (Calamia and Manoil, 1990).

As shown with the ProW protein (Whitley et al., 1994) long periplasmic N-terminal tails fused to reporter proteins could artificially remain cytoplasmic, if the translocation signals are localized in the following transmembrane domain. Therefore, the topological model of KdpD presented in Fig. 7was confirmed not only by the LacZ and PhoA fusion studies, but also by the protease treatment of spheroplasts with and without Triton X-100. Using spheroplasts in the absence of Triton X-100, KdpD was completely resistant to kallikrein digestion. On the other hand, in permeabilized spheroplasts KdpD was no longer protected against kallikrein, because the cytoplasmic regions of the protein carrying potential cleavage sites are now accessible to the protease.

An analysis of the amino acid distribution within the polypeptide chain is also in accord with the model proposed in Fig. 7. The hydrophobic stretch from residues Val-27 to Ala-43 is interrupted by a lysine and is closely followed by two arginines. Since signal sequences are extremely sensitive to the presence of positively charged residues at their C-terminal end (Andersson and von Heijne, 1993), this segment is unlikely to function as a translocation signal. Furthermore, the overall amino acid composition of the N-terminal region (residue 1-400) is more similar to that of a typical cytoplasmic than a periplasmic region (Nakashima and Nishikawa, 1992) as checked with the TOP-PRED program.^3 In addition, the distribution of charged residues may function as a topogenic determinant (Boyd and Beckwith, 1989) and the ``positive-inside'' rule (von Heijne, 1992) is in line with the model of KdpD presented here. All cytoplasmic regions which are at the border of hydrophobic domains possess a net positive charge, whereas the two periplasmic loops possess a net negative or no net charge at all (Fig. 7).

A final argument in favor of the model presented stems from the analysis of a mutant strain, in which the four membrane-spanning segments in the middle of KdpD have been deleted. The truncated protein is still associated with, but can be removed from the membrane by treatment with 2 M urea. This observation lends support to the notion that the N- and C-terminal regions are both cytoplasmic.

Induction of the Kdp system occurs when the K concentration of the medium becomes growth-limiting or if the turgor pressure decreases (Altendorf et al., 1992). Based on the topological model presented, it is tempting to speculate that the four membrane-spanning helices of KdpD could sense ``turgor'' as changes in the stretch of the plane of the membrane, whereas the cytoplasmic N-terminal domain could be a sensor of the ionic conditions in the cytoplasm, which may modulate the primary signal. Experiments are under way to test this intriguing hypothesis.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 171/B5) and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg).

To whom correspondence should be addressed: Universität Osnabrück, Fachbereich, Biologie/Chemie, D-49069 Osnabrück, Germany. Tel.: 01149-541-969-2864; Fax: 01149-541-969-2870.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); AP, alkaline phosphatase.

(^2)
W. Puppe, P. Zimmann, and K. Altendorf, submitted for publication.

(^3)
G. von Heijne, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. G. von Heijne (Stockholm University) for interpretations of the data obtained with the TOP-PRED program for KdpD, Dr. M. Ehrmann (University of Konstanz) for suggesting the use of kallikrein and for other useful information, Dr. G. Deckers-Hebestreit for critically reading the manuscript, H. Gerdes for technical assistance, and J. Petzold for typing the manuscript.


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