(Received for publication, December 1, 1995)
From the
OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes. Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli. The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons. Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm. Expression of OxlT in E. coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate. The unusually high velocity of OxlT-mediated transport is also preserved in E. coli. We conclude that the essential features of OxlT are retained on its expression in E. coli.
The Gram-negative anaerobe, Oxalobacter formigenes, derives metabolic energy from the decarboxylation of oxalate (1, 2) by using a ``proton-motive metabolic cycle''(3, 4) . In O. formigenes, which provided the first case study of such a proton-motive cycle(3, 4) , entry of divalent oxalate is coupled to the exit of its decarboxylation product, monovalent formate, leading to formation of an internally negative membrane potential. Because intracellular oxalate decarboxylation consumes a cytosolic proton, entry of negative charge is accompanied in stoichiometric fashion by appearance of internal hydroxyl ion. As a result, the combined activities of the vectorial antiport reaction and the scalar decarboxylation step comprise a thermodynamic proton pump(3, 4) . In this way, O. formigenes establishes the proton-motive force required for both the synthesis of ATP by reversal of a dicyclohexylcarbodiimide-sensitive ATPase (29) and for the support of other membrane reactions requiring a proton-motive force.
Early experiments based on
reconstitution of activity from crude detergent extracts suggested the
oxalate/formate exchange reaction is mediated by a membrane
carrier(3) . This reasoning was strengthened by finding that
oxalate transport is catalyzed by a single protein, OxlT, whose
SDS-PAGE ()mobility (
38 kDa) resembles that of other
bacterial carrier proteins(5) . However, it was not possible to
complete the argument by examination of the OxlT amino acid sequence.
For this reason, the work described here was directed to the cloning
and sequencing of OxlT. An additional objective was to determine
whether OxlT function is retained after expression in Escherichia
coli. If so, future studies of this unusual antiporter could
exploit the advantages of a genetically tractable host. The work
described here indicates that the amino acid sequence of OxlT conforms
to the general pattern found for most membrane carriers, including the
presence of twelve likely transmembrane segments. Functional studies
further suggest that the main properties of OxlT, including its
exceptionally high velocity(3, 5) , are preserved on
its expression in E. coli.
Figure 2: OxlT DNA and amino acid sequences. The DNA sequence encoding OxlT is shown. The corresponding amino acid sequence (single-letter code) is also given, with underlining to indicate predicted transmembrane segments (see Fig. 3).
Figure 3:
Hydropathy profile and topological model
of OxlT. Top, proposed topological model of OxlT derived from
an analysis of hydropathy (bottom) and from consideration of
the distribution of charged residues (see text). Individual amino acids
are not indicated as such (see Fig. 2). Instead, negatively
charged residues (Asp and Glu) are shown as gray squares, and
except for Lys, positively charged residues (Arg and Lys)
are given as solid circles. Enlarged circles show the
expected locations of Cys
, Cys
, and
Lys
. Bottom, hydropathy profile of the OxlT
amino acid sequence, performed according to Kyte and Doolittle (20) using a window of 13 residues. Transmembrane segments
1-12 are indicated.
OxlT transport activity was monitored by reconstitution
of protein into proteoliposomes(3, 15, 16) .
In a final volume of 250 µl, 50-100 µl of a detergent
extract was mixed with 1.36 mg of bath-sonicated liposomes, additional
detergent (to 1.25%), and either 50 mM MOPS/K or 50 mM MOPS/NMG (pH 7). After incubation at 4 °C for 20 min,
proteoliposomes were formed at 23 °C by the addition of 5 ml of a
dilution and loading buffer (pH 7). For estimates of oxalate
self-exchange (Table 1), the loading buffer contained 100 mM potassium oxalate, 50 mM MOPS/K, and 1 mM dithiothreitol. To assess oxalate/formate exchange (see Fig. 5), the loading buffer was either 100 mM potassium
formate or 100 mM NMG formate, along with 50 mM MOPS/K or 50 mM MOPS/NMG and 1 mM dithiothreitol. Formation of proteoliposomes was complete within
20 min, at which point we used one of two protocols to assess OxlT
activity. In a rapid filtration assay (17) to monitor oxalate
self-exchange (Table 1), 0.2 ml of the proteoliposomal suspension
was applied directly, under vacuum, to the center of a 0.22-µm GSTF
Millipore filter. The external medium was removed by two 5-ml rinses
with assay buffer (100 mM KSO
and 50
mM MOPS/K, pH 7), and on release of the vacuum the assay began
as proteoliposomes were covered with 0.25 ml of assay buffer containing
100 µM [
C]oxalate. The reaction was
terminated 3 min later by filtration and three quick rinses with assay
buffer. Alternatively (see Fig. 5), formate-loaded
proteoliposomes were isolated by centrifugation (16) and
resuspended in a small volume of their K- or NMG-based loading buffers.
Subsequently, they were diluted 120-fold into either NMG- or K-based
assay buffers, as above, containing 100 µM [
C]oxalate, with or without 1 µM valinomycin. In this way, it was possible to generate a membrane
potential whose polarity was either interior positive (potassium
outside, NMG inside) or interior negative (NMG outside, potassium
inside). As an additional basis for comparison, proteoliposomes were
loaded with NMG-formate and tested using the NMG-based assay buffer.
Figure 5:
OxlT expressed in E. coli catalyzes the electrogenic exchange of formate and oxalate. The
detergent extract of IPTG-induced cells carrying pBluescript II
SK+ ( Fig. 4legend) was used to prepare proteoliposomes (or
liposomes) loaded with potassium formate or NMG formate, as described
under ``Experimental Procedures.'' To begin the reaction,
proteoliposomes (or liposomes) were diluted into NMG- or
potassium-based assay medium (as shown) containing 100 µM [C]oxalate with 1 µM valinomycin or the equivalent amount of carrier ethanol as shown.
Samples were taken for filtration and washing at the indicated times.
The presence of external potassium (K
) or
internal potassium (K
) is indicated on the graph.
Proteoliposomes (and liposomes) loaded and assayed using only NMG-based
solutions were also tested, but these data have been omitted for
clarity. Transport by these particles (with or without valinomycin) was
essentially identical in rate and extent to the ionophore-untreated
controls shown here.
Figure 4: Expression of OxlT in E. coli. IPTG-induced and uninduced XL3 cells carrying pBluescript II SK+ or the OxlT expression vector, pBKOxlTSK+, (see Table 1) were harvested. Expression of OxlT was monitored by an immunoblot using antibody directed against the OxlT N terminus (see ``Experimental Procedures''). Lane 1, mass standards, as indicated; lane 2, 14 µg of uninduced cells (pBluescript II SK+); lane 3, 14 µg of uninduced cells (pBKOxlTSK+); lane 4, 14 µg of IPTG-induced cells (pBluescript II SK+); lane 5, 14 µg of IPTG-induced cells (pBKOxlTSK+); lane 6, 2.5 µg of O. formigenes membrane vesicles. Detergent extracts prepared from these cells were used in the experiments of Table 1and Fig. 5.
Figure 1: OxlT N-terminal sequences. The N-terminal sequence determined by microsequencing of purified OxlT (top) is compared with the N-terminal sequence specified by the cloned gene, oxlT (bottom).
The DNA sequence of the gene, oxlT, indicates that expression of its encoded protein (OxlT) follows patterns well established for bacterial systems. Thus, a likely promoter having -35 and -10 sequences of TTGAAA and TTCAAT, respectively, occupies a 29-base interval ending 70 nucleotides upstream of the initiating codon, AUG. Transcriptional termination is probably mediated by a 31-base stem-loop structure (AAAAAAGCCCGGCTTTCCGCCGGGCTTTTTT) that begins 72 nucleotides from the first of two in-frame stop (UAA) codons.
Analysis of OxlT hydropathy according to
the method of Kyte and Doolittle (20) (Fig. 3) suggests
the presence of 12 hydrophobic segments, each of sufficient length to
constitute a transmembrane -helix (TM1-12). A similar
analysis according to Rost et al.(21) predicts 11
transmembrane
-helices, including TM1 and TM3-12 (Fig. 3) but excluding TM2, whose peak hydropathy value is the
lowest of the 12 putative transmembrane segments (Fig. 3).
Although membrane carriers with 11 transmembrane segments have been
described in bacteria(22, 23) , it is more typical to
find examples with 10 or 12 transmembrane
regions(4, 23, 24) . For this reason, our
initial model of OxlT topology (Fig. 3) assumes the 12
transmembrane segments suggested by analysis of hydropathy. This
initial model also conforms to the common finding (4, 23, 24) of a central cytoplasmic loop
that separates the regions containing TM1-6 and TM7-12.
To orient the proposed OxlT structure with respect to cytoplasmic
and extracellular phases, we used the observation of von Heijne (25) that transmembrane segments often have an excess of
positively charged residues at their cytoplasmic ends, especially in
bacterial systems. It is evident that in our proposed structure (Fig. 3, top), charged residues are assigned to either
the extracellular (net charge of -1) or cytoplasmic (net charge
of +13) surfaces, with the exception of the single lysine residue
(Lys) that appears within TM11 (Fig. 3).
The
experiments described in Fig. 4and 5 document that the gene
identified as oxlT specifies the OxlT transport protein and
that the main features of OxlT function are retained in E.
coli. Thus, antibody directed against the OxlT N terminus reported
expression of OxlT in IPTG-induced cells carrying pBKOxlTSK+ but
not in uninduced cells or in cells carrying the parent pBluescript II
SK+ (with or without IPTG) (Fig. 4). It is also evident
that SDS-PAGE profile of OxlT when expressed in E. coli resembles that of authentic OxlT, from O. formigenes,
including the presence of both monomeric (35 kDa) and dimeric
(
65 kDa) forms of the protein (Fig. 4)(5) .
Equally important, in this same experiment we showed that appearance
of OxlT immunoreactivity coincides with acquisition by induced cells of
a capacity to catalyze both the oxalate self-exchange reaction and the
electrogenic exchange of oxalate and formate. For such functional
tests, we prepared detergent extracts from both induced and uninduced
cells (Fig. 4). To examine oxalate self-exchange, oxalate-loaded
proteoliposomes were washed free of external substrate by filtration on
Millipore filters (0.22-µm pore size), and then, while still
affixed to the filters, they were covered for 3 min with an assay
medium containing 100 µM [C]oxalate, followed by a final filtration
and wash. This test (Table 1) gave no indication of oxalate
transport by cells bearing pBluescript II SK+ (± IPTG)
(0.02 µmol/mg protein). By contrast, uninduced cells with
pBKOxlTSK+ displayed a low but significantly positive signal (0.14
µmol/mg protein), whereas IPTG induction led to markedly increased
accumulation of label (2.3 µmol/mg protein) (Table 1).
The
detergent extract from IPTG-induced cells was also used to prepare
proteoliposomes loaded with the potassium or NMG salts of formate so as
to monitor the exchange of oxalate with formate (Fig. 5). The
particles were diluted into media containing NMG sulfate or potassium
sulfate, respectively, so that addition of valinomycin established an
electrical gradient, internally negative or positive in polarity. Such
trials were unambiguous in their findings: imposition of an internally
positive electrical potential strongly stimulated the oxalate transport
observed in controls not treated with the ionophore, whereas imposition
of an internally negative potential completely inhibited the reaction.
Proteoliposomes prepared and assayed in the absence of potassium were
unaffected by valinomycin and showed oxalate transport virtually
identical to that found for the potassium- or NMG-loaded
proteoliposomes not exposed to valinomycin ( Fig. 5legend).
Similar findings had been reported earlier (3, 5) for
OxlT reconstituted from O. formigenes. In both instances, the
pattern of response indicates that the exchange of oxalate and formate
is electrogenic, with negative charge moving in parallel with oxalate.
Because the pK's for oxalate are 1.23 and
3.83, the simplest model is that the OxlT transporter, whether
expressed in O. formigenes or E. coli, mediates
exchange of divalent oxalate and monovalent formate.
The work summarized here had as its main goal the cloning and sequencing of OxlT, the oxalate/formate antiport protein of O. formigenes. Several criteria show this goal has been met. In particular, the cloned gene specifies the N-terminal sequence found in authentic OxlT (Fig. 1), and expression of this gene confers upon E. coli the capacity to mediate both the homologous self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate ( Table 1and Fig. 5). We therefore conclude that this antiport protein retains its most important functional properties when expressed in E. coli. It is likely the main physical characteristics of OxlT are also preserved in E. coli, because the OxlT SDS-PAGE profiles in E. coli and O. formigenes are equivalent (Fig. 4) and because the positive response to an N-terminal peptide-directed antibody suggests OxlT retains its natural N terminus (Fig. 4).
Analysis of the OxlT amino acid sequence reveals a polytopic hydrophobic protein (Fig. 3) whose general structure resembles that of known membrane carriers in the several respects(4, 22, 24) : (i) the presence of 12 (or 11) presumed transmembrane segments; (ii) N- and C-terminal regions facing the cytoplasm (presuming an even number of transmembrane segments); (iii) the finding of a cytoplasmic loop midway along the sequence (residues 190-219), separating the region containing TM1-6 from that containing TM7-12; and (iv) an excess of positively charged residues at the presumed cytoplasmic surface. Although there is no apparent sequence homology between OxlT and known membrane carriers (or other transporters), these general features, along with the earlier biochemical characterization, are sufficient to classify OxlT as a conventional secondary transport protein.
The
OxlT predicted structure has two additional features deserving of
specific comment. First, we note the presence of a single charged
residue (Lys) within TM11 (Fig. 3, top).
Because OxlT substrates are anionic (oxalate
and
formate
), the presence of this apparently
uncompensated positive charge in the hydrophobic sector prompts the
hypothesis that Lys
forms part of an anionic binding
center within the substrate translocation pathway. Preliminary tests
are compatible with this idea, because several uncharged substitutions
at position 355 give variants that fail to transport, whereas the K355R
derivative retains activity. (
)A second finding of interest
is that OxlT has only two cysteine residues (Cys
and
Cys
). Because neither of these cysteines is required for
function,
OxlT presents an attractive target for cysteine
scanning mutagenesis, an approach that has proven valuable to the study
of several membrane transport
systems(26, 27, 28) .
Evaluation of
oxalate transport ( Table 1and Fig. 5) supports the idea
that the main features of OxlT selectivity are retained in E.
coli. Moreover, calculations using these data suggest that the
unusually high velocity of OxlT is also preserved in this expression
system. Thus, detergent extracts from induced E. coli yielded
a potential-stimulated oxalate/formate antiport rate of 24
µmol/min/mg protein (Fig. 5), whereas for the same
conditions we found an exchange rate of 16 µmol/min/mg protein
using O. formigenes(3) . And in further work (not
given), we found the kinetic parameters of oxalate self-exchange to be
the same in E. coli and O. formigenes(3) (Michaelis constants of 0.2 ± 0.03 versus 0.24 mM, and maximal velocities of 102 ± 7 versus 99 µmol/min/mg protein, respectively). Because
immunoblots gave about equal staining for nearly equivalent amounts of E. coli or O. formigenes membrane protein (see Fig. 4legend), it appears that OxlT is expressed at comparable
levels in the two cell types. Accordingly, the specific activity of
OxlT is largely unaffected by expression in E. coli. We note
that the transport rates observed in crude extracts from E. coli (20-100 µmol/min/mg protein) are unusually high but
that this is anticipated for OxlT, which has the highest known maximal
velocity among carriers of organic substrates (5) . That this
feature, too, is preserved in E. coli both confirms early work
and suggests the value of further study in an environment, such as E. coli, conducive to both biochemical and genetic
manipulations.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40075[GenBank].