(Received for publication, May 30, 1995; and in revised form, August 21, 1995)
From the
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.
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.
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.
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-
-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-
-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.
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--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
). 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
HPO
, and cell
debris were removed by centrifugation for 10 min at 13,000
g before measuring the absorbance at 420 nm. In both cases
units of enzyme activity are defined as OD
1000/t
v
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.
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.
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.
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.
It has already been
well established that the alkaline phosphatase is inactive in the
cytoplasm (Manoil et al., 1990), whereas the
-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
-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
-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 -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).
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.
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 -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
-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
-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. 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.