Abteilung Mikrobiologie, Barbarastraße 11, Universität Osnabrück, D-49069 Osnabrück, Germany1
Laboratory of Molecular Cell Biology, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263, Japan2
Author for correspondence: Evert P. Bakker. Tel: +49 541 969 3515. Fax: +49 541 969 2870. e-mail: Bakker_E{at}biologie.uni-osnabrueck.de
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ABSTRACT |
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Keywords: ABC system, peptide transporter family, K+ transport, Kir-SUR system, regulation by adenine nucleotides
Abbreviations: ABC, ATP-binding cassette; EcTrk, VaTrk, Trk from E. coli and V. alginolyticus, respectively; EcSap and Ecsap, Sap proteins and sap genes, respectively, from E. coli; pmf, proton-motive force; VaSap and Vasap, Sap proteins and sap genes, respectively, from V. alginolyticus
The accession numbers for the nucleotide sequences reported in this paper are X97282 and AB015765.
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INTRODUCTION |
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The trkA gene product (TrkA) is required for the activity of the Trk systems from E. coli and Salmonella typhimurium (Dosch et al., 1991 ; Stumpe et al., 1996
; Parra-Lopez et al., 1994
). It occurs in many prokaryotes, including Vibrio alginolyticus (Nakamura et al., 1994
; 1998a
), and several archaea (Stumpe et al., 1996
; Durell et al., 1999
). TrkA is a peripheral membrane protein, attached via TrkH or TrkG to the inner side of the cytoplasmic membrane (Bossemeyer et al., 1989
; Parra-Lopez et al., 1994
; Nakamura et al., 1998a
). TrkA from E. coli (EcTrkA) contains two putative NAD+-binding sites, but although both NAD+ and NADH have been shown to bind to the isolated protein (Schlösser et al., 1993
), it is not known whether these dinucleotides play a role in K+ transport via the Trk system in vivo (Stumpe et al., 1996
).
Little is known about the function of trkE. TrkH requires trkE, but TrkG shows residual activity in the absence of a functional trkE gene (Dosch et al., 1991 ). Moreover, V. alginolyticus Trk (VaTrk) is fully active in an E. coli
trkE strain (Nakamura et al., 1998a
). In both S. typhimurium and E. coli trkE maps inside the sapABCDF operon [Parra-Lopez et al. (1993)
and W. Epstein, personal communication, respectively]. This operon encodes an ATP-binding cassette (ABC) transporter of unknown function from the subgroup of peptide-uptake systems (Parra-Lopez et al., 1993
; Linton & Higgins, 1998
). Since the sapABCDF operon is required for the resistance of S. typhimurium towards the small, strongly cationic protein protamine (Groisman et al., 1992
), Groisman and his colleagues have speculated that SapABCDF detoxifies protamine by transporting it into the cytoplasm, where it is supposed to be degraded by proteases (Parra-Lopez et al., 1993
; Groisman, 1994
). However, our studies with E. coli K-12 have shown that the role of sapABCDF with respect to protamine resistance of E. coli lies in its trkE function, i.e. in its role in K+ transport via the Trk systems (Stumpe & Bakker, 1997
; Stumpe et al., 1998
).
The fact that trkE maps inside the sapABCDF operon (Parra-Lopez et al., 1993 ) raises new interest in early observations that K+ transport via the Trk system depends on ATP (Rhoads & Epstein, 1977
). ATP is believed to activate the system (Stewart et al., 1985
), whereas the transmembrane proton-motive force (pmf) may drive the K+ transport process (Stumpe et al., 1996
). Here we address the question of the relationship between trkE and the sapABCDF operon with respect to their role in K+ transport in E. coli. We show that sapD, one of the two operon genes encoding an ATP-binding subunit, is required for K+ uptake. Changing amino acid residues in the Walker A or B box of SapD led in some situations to an almost complete loss of transport activity, suggesting that the ATP-dependence of the Trk systems is conferred via SapD. Finally, the trkE-independent activities of TrkG and VaTrk activities remained ATP-dependent.
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METHODS |
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Nucleotide sequencing.
The nucleotide sequence of the 5·9 kb E. coli chromosomal insert of plasmid pTWE341 was determined by Eurogentec. Other sequences were determined by MWG-Biotech using the chain-termination method of Sanger et al. (1977) . Nucleotide primers were from the latter company.
Detection of sap gene products.
Plasmid-containing mini-cells of strain DK6 were isolated on a sucrose gradient and their Sap proteins were labelled with [35S]methionine and made visible as described by Reeve (1984) .
N-terminal sequence of Sap proteins from E. coli (EcSap).
This sequence was determined for SapA, SapD and SapF, encoded by plasmid pTWE341 or one of its derivatives (Fig. 1a). To this end these proteins were overproduced with the aid of the T7 promoter in front of the sapABCDF operon, the cells were broken by sonication, membranes were collected by ultracentrifugation and membrane proteins were separated by SDS-PAGE. SapA, SapD or SapF were cut out from the gel and their N-terminal sequence was determined by automatic Edman degradation in an Applied Biosystems 473A apparatus.
Transport experiments and ATP content.
All of these experiments were done at 2022 °C and were repeated at least two times. Figs 3, 4
and 6
give mean values of three independent experiments. Figs 5
, 7
, 8
and Table 2
give results from a single experiment, of which all of its elements were repeated at least once, but not necessarily in a single experiment. For the calculation of the cell content of K+, [14C]proline, [14C]glutamine and ATP, a cell suspension with an OD578 of 1·0 was taken to be equivalent to 0·33 mg cell dry wt ml-1 (Bakker & Mangerich, 1981
).
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K+ and energy reserves of cells of atp+ or atpB-C strains were depleted by shaking them for 1·75 h at 37 °C in the presence of 5 mM 2,4-dinitrophenol in minimal mineral growth medium plus 0·15 mM chloramphenicol, but without K+, glucose, thiamine and other antibiotics. The protonophore was removed by washing the cells three times with cold buffer A, containing 75 mM sodium phosphate, 0·4 mM MgSO4 and 0·15 mM chloramphenicol, pH 7·0. Subsequently, cells were suspended in buffer A at a concentration of 1020 mg dry wt ml-1 and kept on ice for up to 3 h before the start of the transport experiment. For uptake experiments of K+ and radioactive compounds cells were suspended at 1 and 0·67 mg dry wt (ml buffer A)-1, respectively. Glucose (10 mM) or 20 mM sodium succinate was added at t=-15 and -30 min, respectively, and the cell suspension was shaken at 160 r.p.m. At t=0 min, 2 mM KCl, 2 µM [14C]glutamine (40 nCi ml-1) or 4 µM [14C]proline (100 nCi ml-1) was added and the suspension was shaken as described above. For the [14C]proline-uptake experiments the buffer contained 2 mM KCl. Samples for cell K+ determination were taken and treated as described above. For the measurement of radioactivity in the cells 0·3 ml samples of the suspension were filtered through 2·5 cm diameter glass fibre GF-5 filters (pore size 0·45 µm; Macherey & Nagel). The cells on the filter were washed twice with 100 mM LiCl solution, the filters were dried and their radioactivity was measured by liquid-scintillation counting. Samples for ATP determinations in the cells were withdrawn from the same suspension as the one used for measuring K+ content of the cells. After cell lysis by cold 5 mM H3PO4 containing 12% perchloric acid, readjustment of the suspension to pH 7·0 with a solution containing 2 M KOH plus 0·3 M MOPS and removal of KClO4 and protein by centrifugation, ATP was determined in the supernatant by using a luminescence assay (Kimmich et al., 1975
).
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RESULTS |
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Since the action of VaTrk in E. coli is independent of the EcsapABCDF operon (Nakamura et al., 1998a ) it was important to determine whether V. alginolyticus also contains a sapABCDF operon. Clones containing most of sapA and complete sapBCDF of this organism were obtained (Fig. 1b
). Their nucleotide sequence was determined and filed under number AB015765 at the DDBJ database. The percentage of identical residues in E. coli and V. alginolyticus varies between 43% for SapA and 64% for SapD. By contrast, the Sap proteins of E. coli and S. typhimurium have highly identical sequences, varying between 90% for SapA, and 96 and 98% for the ATP-binding subunits SapD and SapF, respectively.
sap gene products in mini-cells
To obtain more information on the function of sap genes, deletion plasmids were constructed from plasmids pTWE341, pKT7 and pKT70 (Fig. 1). The Ecsap plasmids, derived from plasmid pTWE341, were brought into cells of the mini-cell-producing E. coli strain DK6 (Klionsky et al., 1984
). These cells made all five plasmid-encoded EcSap proteins (Fig. 2
) under conditions at which the T7 promoter should not be active, suggesting that in this system sap genes are transcribed from their own promoter(s). However, with sapA DNA as a probe in Northern hybridization experiments, it was observed that plasmid pTWE341 made very large transcripts of up to 9 kb (results not shown), indicating that under these conditions sapABCDF transcription was not necessarily directed by its own promoter(s).
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K+ uptake via TrkH in V. alginolyticus (Va) sapBCDF-plasmid containing cells of strain LB625 was stimulated by growing the cells in the presence of 0·3 mM IPTG. Under these conditions maximal TrkH activity was comparable to that of cells containing E. coli sapDF genes on the plasmid (Fig. 3b and 3a
, respectively). VaSapD stimulated TrkH activity to some extent (plasmids pKT72 and pKT77; Fig. 3b
) and VaSapF alone was inactive (pKT701; Fig. 3b
). However, in contrast to the situation with EcSapD, plasmids encoding both of the integral membrane proteins VaSapBC supported the VaSapD function, since the stimulation of K+ uptake by cells containing plasmid pKT7 (VasapBCD+; Fig. 3b
) was larger than that of cells containing either plasmid pKT72 (VasapCD+; Fig. 3b
) or plasmid pKT77 (VasapD+; Fig. 3b
). Finally, only plasmid pKT70, containing VasapBCDF, exerted the maximal effect (Fig. 3b
), suggesting that VaSapF also supports the function of VaSapBCD in this test. Despite the fact that these plasmids directed the synthesis of approximately equal amounts of VaSapD in E. coli mini-cells (results not shown), these effects might still be due to differences in VasapD gene expression by the different plasmids in strain LB625.
K+ transport via TrkG
For these experiments we used strain LB680 (sapABCDF::KmR
trkH::CmR) containing the plasmids in Fig. 1
. The same type of experiments were carried out as in Fig. 3
. The TrkG system exhibited about 30% residual activity in the absence of sap genes (Fig. 4a
), confirming earlier data (Dosch et al., 1991
). However, in contrast to the latter, we observed that this effect was due to a reduction of Vmax for K+ of TrkG (results not shown). With respect to stimulation of K+ uptake by plasmid-encoded EcSap proteins, the results with TrkG differed from those of TrkH, in that (i) plasmid-encoded SapD alone was sufficient for full stimulation of K+ transport, (ii) the presence of plasmid pCH100, encoding SapABCD was without effect (Fig. 4a
) and (iii) the additional presence of SapF gave the full effect (pTWE341; Fig. 4a
).
With one exception, the effects of Vasap plasmids on TrkG were similar to those of their E. coli counterparts (Fig. 4b and 4a
, respectively): the presence of plasmid pKT7 (VasapBCD+) inhibited the intrinsic K+-uptake activity of strain LB680 by as much as 70% (Fig. 4b
). We will return to the results of Figs 3
and 4
in the Discussion.
Protein variants of the EcSapD Walker A box residue Lys-46
SapDF are the ATP-binding cassette (ABC) subunits of the SapABCDF complex and in analogy with other bacterial systems, we assume that through ATP hydrolysis they energize the uptake by the cells of the unknown substrate via the SapABCDF complex (Schneider & Hunke, 1998 ). These ABC subunits contain conserved sequences essential for ATP binding and/or ATP hydrolysis (Walker et al., 1982
). The Walker A box consists of the conserved sequence GXXGXGKT/S (Walker et al., 1982
; Boos & Lucht, 1996
; Schneider & Hunke, 1998
). It reads as 40-GESGSGKS-47 in both EcSapD and VaSapD. In HisP, for which a crystal structure is available (Hung et al., 1998
), the charged group of the side-chain Walker A box lysine residue interacts with the ß-phosphate group of ATP.
To determine the effect of adenine nucleotides via EcSapD on K+ uptake via EcTrkH, the Walker A box lysine codon 46 of sapD in plasmid pCH105 (sapDF+) was replaced by codons for arginine, glutamine, methionine, isoleucine or glycine (plasmids pCC11, pCC7, pCC3, pCC4 and pCC8, respectively) and the effect of these changes on K+ uptake by strain LB625 containing these plasmids was determined (Fig. 5a). Remarkably, in none of these strains was K+ transport activity inhibited completely. Even with plasmid pCC8 (encoding SapD variant Gly-46, which showed the largest effect) K+ uptake still occurred at a rate of about 25% of the control. Only the presence of plasmid pCC101, containing sapDF in which the codon for Lys-46 of sapD had been deleted, hardly showed any stimulation of net K+-uptake activity by strain LB625 above background (Fig. 5a
). This effect is unlikely to be due to lack of sapDF gene expression, since the pCC101-encoded SapD mutein was made in normal amounts in E. coli mini-cells (results not shown).
Similar results (not shown) were obtained with cells that contained both variants of SapD residue Lys-46 and of the similar Walker A box SapF residue Lys-53 [plasmids pCC1 (SapD-46Gln; SapF-53Gln) or pCC2 (SapD-46Arg; SapF-53Gln)] or with cells containing plasmid pCC111, which was derived from pCH106 (only sapD, Fig. 1a), encoding the variant SapD-46Gly: none of these cells showed a complete inhibition of transport activity.
Protein variants of the EcSapD Walker B box residue Asp-183
The Walker B box of ABC proteins consists of the conserved sequence hhhhD, in which h represents an apolar residue and D is an aspartate residue (Walker et al., 1982 ; Boos & Lucht, 1996
; Schneider & Hunke, 1998
). It reads as 179-Leu-Leu-Ile-Ala-Asp-183 in both EcSapD and VaSapD. In HisP the side chain of the conserved Walker B box residue aspartate interacts via a water molecule with both the ß- and
-phosphate group of ATP (Hung et al., 1998
). The residue Asp-183 of SapD in plasmid pCH105 (sapDF+) was replaced by glutamate, asparagine or lysine (plasmids pCC15, pCC16 and pCC17, respectively). The effect of these changes on K+ uptake via TrkH was determined (Fig. 5b
). The strongest inhibition (85%) was observed for the plasmid encoding the SapD variant Lys-183 (Fig. 5b
).
Protein variants in the EcSapD signature motif residues Gly-162 and Gln-165
In ABC proteins the conserved signature motif is thought to play a role in energy coupling between ATP hydrolysis and transport. In both HisP and MalK this region is located relatively far away from the nucleotide-binding site in a region that might interact with the integral membrane proteins of the transport complex (Hung et al., 1998 ; Diederichs et al., 2000
). For MalK from S. typhimurium, Schmees et al. (1999)
have characterized several protein variants of linker region residues that still bind the ATP analogue 8-azido-ATP, but for which the MalEFGK2 complex has become inactive in maltose transport. We observed that the similar signature motif SapD variants Ala-162 (instead of glycine) and Leu-165 (instead of glutamine) exhibited a 20 and 40% inhibition of K+-uptake activity of the TrkH system, respectively (Fig. 5c
).
The most likely explanation for the data in Fig. 5 is that ATP binding to SapD rather than its ATP hydrolysis is required for TrkH activity, confirming an early conclusion that ATP activates the E. coli Trk system (Stewart et al., 1985
).
Trk activity in the absence of sap gene products
Fig. 6 shows that in a
sapABCDF background K+-uptake activity by TrkH activity was low, but not zero as is the case for
trkH
trkG
sapABCDF strain LB690 (Fig. 6
). Hence, both TrkH and TrkG are partially active in a
sapABCDF background (Figs 4
and 6
). To obtain more information on this phenomenon, we investigated the influence of plasmid pAS8-encoded TrkG on K+ transport by strains LB625 (
trkG
sapABCDF), LB680 (
trkH
sapABCDF) and LB690. All three strains took up K+ about twice as rapidly as did strain LB680 without the plasmid (Fig. 6
), indicating that trkG exerts a gene dose effect in this process. As reported previously (Nakamura et al., 1998a
), K+ uptake via plasmid-encoded VaTrk in E. coli was independent of sapABCDF (Fig. 6
) and even exceeded K+-uptake activity by strain LB625(pTWE341) (Fig. 6
). Additional results (not shown) indicated that the differences in transport activity observed in Fig. 6
are due to differences in Vmax values for K+ of the different systems.
Energy coupling to K+ uptake via Trk in E. coli sapABCDF strains
The easiest way to explain the data in Fig. 6 is to assume that K+ uptake via EcTrkG and VaTrk is independent of ATP. Hence, the mode of energy coupling to K+ uptake by these strains was investigated. For this purpose strains LB627 (
atpB-C
sapABCDF
trkG), LB682 (
atpB-C
sapABCDF
trkH) and LB692 (
atpB-C
sapABCDF
trkH
trkG) were constructed by P1 transduction from strains LB625, LB680 and LB690, respectively. In contrast to wild-type strains, ATP levels and the pmf can be influenced independently from each other in an atp (formerly unc) strain. This is achieved by: (i) glucose metabolism in the presence of a protonophore, like 2,4-dinitrophenol, resulting in a high ATP level in the absence of a pmf; (ii) succinate respiration by energy-starved cells, leading to a high pmf and a low ATP level in the cells; and (iii) glucose metabolism in the absence of a protonophore, giving rise to both a high cytoplasmic ATP concentration and a high pmf (Berger, 1973
; Rhoads & Epstein, 1977
). Such experiments on the mode of energy coupling to K+ transport have been carried out with the E. coli Trk system (Rhoads & Epstein, 1977
). Their results suggested that Trk activity depends both on a high pmf and a high cytoplasmic ATP concentration. However, at that time it was not known that in this E. coli strain Trk consists of the two separate TrkH and TrkG systems, each of which contribute approximately equally to total Trk activity (Dosch et al., 1991
; Schlösser et al., 1995
). We determined the mode of energy coupling to K+ transport for both EcTrk systems [strains LB625(pTWE341) and LB627(pTWE341) for TrkH and strains LB680(pTWE341) and LB682(pTWE341) for TrkG], for TrkG in a
sapABCDF background [strains LB680(pAS8) and LB682(pAS8)] and for plasmid-encoded VaTrk in an E. coli
sapABCDF background [strains LB690(pKT65) and LB692(pKT65)]. The results on K+ uptake were compared with transport of proline and glutamine by these cells, which give an indication of the presence of a high membrane potential (which is the main driving force for proline uptake under these conditions) and a high cytoplasmic ATP concentration (ATP is the driving force for glutamine uptake via an E. coli ABC transporter), respectively (Berger, 1973
; Rhoads & Epstein, 1977
). The ATP content of the cells was also measured.
In glucose-metabolizing cells of the atp strain LB627(pTWE341) the dependence of the initial rates of K+ uptake via the TrkH system, of proline uptake and of glutamine uptake were measured as a function of 2,4-dinitrophenol concentration (Fig. 7
). Both the uptake of K+ and that of proline were strongly inhibited by the protonophore under conditions at which both the ATP level in the cells and the rate of glutamine uptake remained high (Fig. 7
). Similar results were obtained with the EcTrkG system [strain LB682(pTWE341)], chromosome and plasmid-encoded TrkG in a
sapABCDF background [strain LB682(pAS8)], and plasmid-encoded VaTrk in an E. coli
sapABCDF background [(strain LB692(pKT65)] (results not shown). These data indicate (i) that K+ uptake via all four systems depends on the membrane potential and (ii) that in contrast to glutamine transport a high cytoplasmic ATP concentration alone is not sufficient to drive K+ transport via these systems.
Next it was investigated whether ATP is also required. Fig. 8 shows such an experiment for EcTrkH. It was observed (i) that the uptake of both K+ and glutamine occurred in the atp wild-type strain LB625(pTWE341) with either glucose or succinate as the substrate; (ii) that these compounds were also taken up by the atp strain LB627(pTWE341) in the presence of glucose, but not in the presence of succinate (Fig. 8a
, c
); (iii) that proline was taken up under all four conditions (Fig. 8d
); and (iv) that succinate-metabolizing cells of the
atp strain were not able to generate a high cytoplasmic ATP level (Fig. 8b
). These data show a positive correlation between TrkH activity and the cytoplasmic ATP concentration. Similar results were obtained with the chromosomally encoded EcTrkG system (Table 2
). Together these data support the notion that the activity of the E. coli Trk system [which is now known to consist of the systems TrkH and TrkG (Dosch et al., 1991
)] requires both a pmf and ATP for activity (Rhoads & Epstein, 1977
; Stewart et al., 1985
).
It was then investigated whether this also holds true for K+ transport via EcTrkG and VaTrk in E. coli sapABCDF strains (Table 2
). The results were very similar to those obtained with E. coli TrkH and TrkG systems (Fig. 8
, Table 2
), suggesting that energy coupling to K+ transport was similar in all four situations. Hence, these data argue against the notion that K+ uptake via Trk systems in a
sapABCDF background in E. coli, as observed in Fig. 6
, occurred due to a lack of ATP dependence of this process.
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DISCUSSION |
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In accordance with what is known about other ABC systems (Schneider & Hunke, 1998 ; Schmees et al., 1999
), we conclude that rather aspecific effects such as observed in Fig. 5
suggest that ATP binding to SapD rather than its hydrolysis by the ABC protein is essential for activity of the TrkH system. More specifically, first, Schmees et al. (1999)
have described exactly such effects for the signature motif region protein variants Gly-137
Ala and Gln-140
Leu of the ATP-binding protein MalK from the maltose-uptake system MalEFGK2 from S. typhimurium: ATP binding to the isolated variant MalK proteins still occurs, but these proteins do not hydrolyse ATP and transport activity of the complex is inhibited. Our result that cells with the similar SapD variants Gly-162
Ala and Gln-165
Leu were still active in K+ transport via TrkH (Fig. 5c
) suggests that ATP still binds to these protein variants and that this binding is sufficient for activity. Second, a common effect of mutations in the Walker A box lysine codon is that ATP hydrolysis, but not ATP binding is impaired. Our data that with these protein variants TrkH activity was only partially inhibited (Fig. 5a
), support the notion that ATP binding to SapD is sufficient for TrkH activity. Finally, many mutations in the conserved Walker B box aspartate codon abolish both ATP hydrolysis and ATP binding. Since some mutations in the sapD Walker B box aspartate codon had relatively large effects on K+ transport (Fig. 5b
), these data suggest that at least ATP binding to SapD is required for TrkH activity. However, it should be realized that all of our experiments have been carried out with intact cells, in which under energized conditions the ATP concentration amounts to 35 mM (Table 2
and Schleyer et al., 1993
). This concentration is three orders of magnitude higher than that used to analyse effects of 8-azido-ATP binding to isolated variants of ABC proteins (Schmees et al., 1999
). Hence, it is difficult to compare the results of Fig. 5
with those of isolated proteins. Nevertheless, our data support the older notion that ATP plays a regulatory role in K+ uptake via Trk (Stewart et al., 1985
).
Kir-SUR is a eukaryotic ABC system in which adenine nucleotide binding to SUR regulates the activity of the small K+ channel Kir (Bryan & Aguilar-Bryan, 1999 ). SUR contains two membrane domains equivalent to the integral membrane protein subunits SapB and SapC from SapABCDF and these domains are thought to confer adenine nucleotide dependence to the channel (Bryan & Aguilar-Bryan, 1999
). Kir-SUR resembles Sap-Trk in the following aspect: Kir contains only two transmembrane helices and thereby resembles the bacterial K+ channel KcsA. The Kir active complex is thought to be a tetramer surrounded by four SUR subunits, forming a huge supra-molecular membrane complex (Shyng & Nichols, 1997
; Inagaki et al., 1997
; Clement et al., 1997
; Babenko et al., 1998
). The main part of the TrkH protein consists of a domain composed of eight transmembrane helices that is likely to have evolved from four covalently linked K+-channel subunits of the KcsA/Kir type (Durell et al., 1999
; Durell & Guy, 1999
). By analogy with the Kir-SUR complex we would have expected the Sap-TrkH complex to contain at least the SapBCDF proteins, and that four SapB or SapC proteins interact with one TrkH or TrkG subunit by helixhelix interaction in the apolar part of the membrane. However, we have found no direct evidence for this, since most of our results with plasmid-encoded Sap proteins from E. coli and V. alginolyticus suggest that SapD is sufficient for K+ transport via the EcTrk systems (Figs 3
and 4
). The fact that some of these data indicate that other subunits than SapD also support the activity of the Trk systems is most easily explained by assuming differences in sapD gene expression in the different cells.
The best interpretation of the observation that TrkG and VaTrk are active in an ATP-dependent manner in E. coli sapABCDF strains (Figs 68
, Table 2
) is to assume that these Trk systems can also interact with an ABC protein other than SapD. This would also explain the apparent contradiction that TrkH systems are widely spread among prokaryotes (Durell et al., 1999
), but that sapABCDF operons are limited to a the
-proteobacteria (results of our genome analyses for Sap proteins are not shown). The ATP dependence of Trk systems from organisms outside this group [e.g. Enterococcus hirae (Bakker & Harold, 1980
)] would then be conferred by interaction with an unknown ABC protein.
An alternative interpretation of these data was also considered. One relatively simple explanation would be that EcTrkAG and VaTrkAH contain an additional adenine nucleotide binding site and that binding of ATP to this site stimulates K+ transport. Such a model would once again be similar to that of the K+ channel Kir, the activity of which has been shown to be inhibited by ATP in the absence of SUR and which has been shown to bind 8-azido-ATP (reviewed by Ueda et al., 1999 ). Isolated TrkA binds dinucleotides rather than ATP (Schlösser et al., 1993
), leaving TrkH/TrkG as the subunits that should bind the nucleotide. However, at present it is not known whether these subunits contain such a site. Moreover, this model presents difficulties in explaining why TrkH and TrkG exert such a different K+ transport activity in a
sapABCDF background. Therefore, we prefer the above explanation that Trk systems can interact with an ABC protein other than SapD.
It remains unclear what the other function of SapABCDF is besides its role in K+ uptake in E. coli. Our preliminary results indicate that sap transcripts are made in cells grown in a mineral salt medium and that neither the presence of oligopeptides, as present in tryptone, nor treatment of the cells with protamine significantly influences the amount of transcript. Moreover, the peptide-uptake subgroup of bacterial ABC systems, to which SapABCDF belongs, covers systems with a broad substrate specificity, including a Ni2+ transporter (Navarro et al., 1993 ) and a haem-binding subunit from Haemophilus influenzae (Hanson et al., 1992
). Hence it is impossible to predict from primary structure data what type of substrate is transported by SapABCDF.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 April 2001;
revised 2 July 2001;
accepted 18 July 2001.
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