From the Department of Physiology, The Johns Hopkins University Medical School, Baltimore, Maryland 21205
Received for publication, September 13, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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OxlT, the oxalate:formate antiporter of
Oxalobacter formigenes, has a lone charged residue, lysine
355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because
Lys-355 is the only charged residue in the hydrophobic sector, we
tested the hypothesis that lysine 355 contributes to the binding site
for the anionic substrate, oxalate. This idea was supported by
mutational analysis, which showed that of five variants studied
(Lys-355 In the anaerobic bacterium, Oxalobacter formigenes, the
proton-motive force is generated by the combined action of an internal oxalate decarboxylation system and the electrogenic
oxalate2 Hydropathy analysis of the OxlT amino acid sequence (3), together with
circular dichroism spectroscopy of the purified, solubilized protein
(8), suggests the presence of 12 transmembrane The experiments reported here sought to address such issues by asking
if one or more residues on TM11 is involved in substrate binding and/or
translocation. To do this, we first examined the role of Lys-355 itself
by analysis of site-specific mutants. We then exploited a panel of
single-cysteine variants to search throughout TM11 for perturbations
attributable to either cysteine substitution or to site-specific
chemical modification. Together, these two lines of study provide
evidence justifying the idea that as substrate passes through OxlT, it
encounters residue(s) along TM11 in the vicinity of Lys-355.
Mutagenesis and Protein Expression--
Wild type OxlT was
encoded within a 1.4-kilobase XbaI-HindIII
fragment in pBluescript II SK+; expression of OxlT was regulated by the
lac promoter (3). This plasmid also served as the vehicle for site-directed mutagenesis using a double-stranded protocol (CameleonTM, Stratagene) to generate mutants of Lys-355.
Similar techniques were used to construct a fully active cysteine-less variant with nine tandem histidine residues added at its C terminus to
facilitate protein purification (8, 10). This latter derivative was
used as the template for generation of the panel of single-cysteine derivatives spanning TM11 (10). OxlT and its mutants were carried in
Escherichia coli strain XL-1, together with plasmid pMS421 (Specr, LacIq) to limit inappropriate basal
expression (3). OxlT expression was induced by addition of 0.5 mM isopropyl-1-thio- Functional Reconstitution and Assays of Oxalate
Transport--
Membrane ghosts, prepared as described (10, 14), were
suspended in distilled water; membrane proteins were solubilized by
incubating for 20 min on ice in Buffer A (20 mM MOPS/K (pH 7), 20% (v/v) glycerol, 4.2 mg/ml E. coli phospholipid, 10 mM potassium oxalate, and 1.5% (w/v) octylglucoside).
Here, and in all work involving thiol labeling (below), the E. coli lipid used for solubilization and reconstitution had been
hydrated with distilled water rather than with the usual 2 mM
In most cases, [14C]oxalate transport was measured by a
rapid filtration assay. Triplicate 100-200-µl aliquots of
proteoliposomes were placed at the center of Millipore GSTF filters
(0.22-µm pore size), washed twice with 5 ml of Buffer B (100 mM potassium sulfate, 50 mM MOPS/K, (pH 7) to
remove external (unlabeled) oxalate, and after interrupting the vacuum,
proteoliposomes trapped on the filter were overlaid with 300 µl of
Buffer B containing 100 µM [14C]oxalate.
Unless otherwise indicated, the assay was terminated after 20 s by
filtration, followed with two washes using 5 ml of iced Buffer B. Depending on experimental design, oxalate transport is given as a
fraction of the maximum incorporation or as relative to that of the
cysteine-less parental protein, after correction for differences in
protein expression (10). This assay provides a wider dynamic range than
available earlier (3, 8, 10) and is especially suited to the parallel
study of a large number of samples.
[14C]Oxalate transport was also monitored by a
traditional filtration assay (1, 3). In these cases, washed
proteoliposomes suspended as a concentrated stock in Buffer B were
diluted 10-fold into this same buffer at 23 °C, and the transport
reaction was initiated by addition of either 100 µM or 1 mM labeled substrate, as specified. At timed intervals,
samples were withdrawn for filtration and washing.
Thiol Labeling--
To screen for thiol reactivity in the TM11
panel of single-cysteine derivatives, membrane ghosts were placed with
excess probe (2 mM MTSCE or 1 mM MTSET) in 20 mM potassium phosphate (pH 8) for 1 h at 23 °C.
Unreacted probe was removed either by a quench using 5 mM
OxlT Orientation after Reconstitution--
The orientation of
OxlT in proteoliposomes was deduced from the pattern of thiol labeling
of cysteines placed at either end of TM11 and also by monitoring the
effects on substrate transport of external trypsin. For thiol labeling,
the target protein (A345C or A370C) was purified (>95%) by
Ni2+-nitrilotriacetic acid affinity chromatography,
essentially as described (8, 10) except that the final elution used a
buffer containing 100 mM potassium oxalate, 50 mM potassium acetate, 20% glycerol (v/v), and 5 mg/ml
diheptanoylphosphatidylcholine (pH 4.5). Prior to reconstitution, 10 µl of the eluate (5-10 µg of protein), was mixed with 240 µl of
Buffer A in which lipid was increased to 10 mg/ml to compensate for
that normally contributed by native membranes (see above). Protein
concentration (40 µg/ml) was close to that of OxlT in crude extracts,
which contained the transporter at 3-5% of membrane protein (8, 10).
After reconstitution, proteoliposomes were washed and finally suspended
in 0.5 ml of Buffer B. Cysteines exposed to the suspending medium
were blocked by exposure to 2 mM MTSET for 15 min at
23 °C. The probe was removed by a 40-fold dilution with iced Buffer
B, followed by centrifugation and resuspension of proteoliposomes to
their original volume in Buffer B. MTSET-pretreated proteoliposomes,
along with untreated controls, were then incubated with either 400 µM MPB or an equivalent volume of its dimethyl sulfoxide
solvent for 60 min at 23 °C. This second reaction was quenched by
dilution, centrifugation, and washing, as above. In some cases, MPB
labeling was followed by addition of trypsin (1 mg/ml) and further
incubation for 90 min at 33 °C, after which protease action was
quenched by dilution, centrifugation, and resuspension (as above) using
iced Buffer B containing 1 mM AEBSF. After
SDS-polyacrylamide gel electrophoresis and after transfer to
nitrocellulose, the presence of MPB was detected by enhanced
chemiluminescence (Pierce, SuperSignalTM) using a horseradish
peroxidase-linked anti-biotin antibody (New England Biolabs). To
monitor recovery of OxlT, the blot was stripped and reprobed with a
polyclonal antibody reactive to the OxlT N terminus (2, 3). Signals
were captured by an automated documentation system (Fuji Medical
Systems, Inc.).
The sensitivity of OxlT to external trypsin was monitored using the
wild-type protein purified as described (8, 10). After reconstitution
(see above), proteoliposomes were placed in 1 ml of Buffer B with and
without trypsin (1 mg/ml) at 33 °C, as described (16). At timed
intervals, samples were diluted 25-fold with iced Buffer B containing
0.5 mM PMSF. After centrifugation and resuspension, as
above, initial rates of [14C]oxalate transport were
determined using the traditional filtration assay with 100 µM labeled substrate.
Modification of S359C by MTS-linked Probes--
Membrane ghosts
prepared using the S359C mutant were extracted and crude extracts were
reconstituted as noted above. To characterize the concentration
dependence of inhibition by MTS-linked probes, proteoliposomes trapped
on a Millipore GSTF filter (0.22-µm pore size) were washed twice with
Buffer B and then overlaid (in triplicate) for 5 min with 0.5 ml of
Buffer B containing MTSCE, MTSES, or MTSET at the specified
concentrations. When protection by substrate was monitored, the 5-min
incubation was in the presence of probe and increasing concentrations
of potassium oxalate (from 0 to 100 mM); a reciprocal
decrease in potassium sulfate (from 100 to 0 mM) served to
maintain constant ionic and osmotic strength. Reactions were terminated
by rapid filtration, followed by two washes with Buffer B, and residual
OxlT function was monitored by a second overlay with labeled substrate,
as noted above. Preliminary trials showed that half-maximal inhibition
of OxlT required 10-30 µM MTSCE, and that nearly
complete inhibition (
Because inhibition by MTSCE occurs on a time scale of minutes,
while turnover for OxlT-mediated exchange takes place in milliseconds or less (1, 2, 8), we also assumed rapid equilibrium between OxlT and
its substrate, as described in earlier kinetic schemes (7, 8). With
these assumptions, the model makes the following quantitative
prediction concerning the parameter F, the fraction of the
OxlT population that remains unmodified by MTSCE.
Chemicals--
Purified E. coli phospholipid was
obtained as a lyophilized powder from Avanti Polar Lipids, Inc. MTSCE,
MTSET, and MTSES were from Toronto Research Chemicals, Inc., and MPB
was from Molecular Probes, Inc. Roche-Calbiochem provided
octylglucoside, while the trypsin (tosylphenylalanyl chloromethyl
ketone-treated) was from Worthington. PMSF and AEBSF were from Sigma.
[14C]Oxalate was from PerkinElmer Life Sciences.
Two classes of experiments explored the idea that Lys-355 and
other residues on TM11 might be associated with substrate binding and
translocation. In one group of studies, described immediately below,
the role of Lys-355 itself was examined by noting the behavior of its
mutants. In a second group of experiments, cysteine-scanning mutagenesis identified a residue, near to Lys-355, which served as an
informative target for cysteine-directed agents. Both lines of study
provided evidence consistent with the idea that Lys-355 and residues in
its vicinity normally serve to aid in the translocation of OxlT substrates.
Mutations of Lys-355 Disrupt OxlT Function--
If Lys-355 is
essential to OxlT function, substitutions at this position should
compromise activity. To test this prediction, we used site-directed
mutagenesis to replace Lys-355 with alternate residues, including
cysteine, glycine, glutamine, arginine, and threonine. In the usual
assays of transport by OxlT (see "Experimental Procedures"), these
mutants displayed little or no function. In one case (K355G) this was
attributable to lack of expression; all other mutants were found in
membranes in amounts comparable to the wild type (data not shown; see
Ref. 10), so that lack of function was due to failure of OxlT itself.
We considered the possibility that such null responses might reflect a
poor substrate affinity, and for that reason we also measured transport
by oxalate-loaded proteoliposomes using external
[14C]oxalate at 1 mM rather than the usual
0.1 mM. In only one case (the K355R variant) did this added
test reveal a significant activity (Fig.
1), although the time course of transport
by the mutant was greatly extended relative to that of the parental
wild type protein. In separate experiments, we performed a further,
kinetic analysis of the K355R derivative. That work showed this mutant to have both an elevated Michaelis constant (Km of
5.4 versus 0.15 mM) and a reduced maximal
velocity (Vmax of 66 versus 4,700 nmol/min/mg protein), leading to a 2,600-fold reduction in catalytic
efficiency (Vmax/Km) relative
to the parental protein. Accordingly, because mutants of Lys-355
retained little or no function, we conclude this residue is essential
for normal OxlT function.
Functionally Significant Residues in TM11--
The behavior of
Lys-355 mutants (Fig. 1) suggests that OxlT requires (at least) the
presence of positive charge at position 355. To ask whether nearby
residues might also have functional significance, we analyzed a panel
of TM11 single-cysteine variants that had earlier been used to
establish topological relationships in this region (10). We first
tested [14C]oxalate transport under initial rate
conditions during the oxalate self-exchange reaction (Fig.
2A). The N347C, A368C, and
I369C variants were too poorly expressed (
In earlier work with this same set of single-cysteine variants, we
showed that the TM11 core region (positions 351-361; Fig. 2A) is inaccessible to Oregon Green maleimide, a hydrophilic
thiol-reactive agent of moderate size (~500 daltons) (10). The
present work shows that, although cysteine substitutions giving reduced
function ( Accessibility to MTSCE and MTSET--
The high velocity of
OxlT-mediated reactions ensures that even cysteine substitution mutants
with low residual activity (Fig. 2A) show satisfactory
signal-to-noise ratios. For this reason we pursued a study of these
variants using the thiol-specific probes, MTSCE and MTSET. These agents
were chosen for their specificity in modification of cysteine (17),
because their polar and linear character increases the probability they
might have access to regions within OxlT near a substrate binding
region (e.g. possibly near Lys-355), and because they
generate reaction products of a clearly distinct character-MTSET
implants a fixed positive charge (RS-SCH2CH2N(CH3)
Membranes containing TM11 single-cysteine variants were exposed to
excess MTSCE or MTSET, with and without tests for reversibility by
later exposure to
Modification of the four peripheral residues (S344C, S345C, G366C,
A370C) might have been predicted, since earlier work characterized these regions as accessible to (and reactive with) Oregon Green maleimide or rhodamine maleimide (Ref. 10, and experiments not shown).
On the other hand, prominent and readily reversible effects at S359C
were unexpected, since this position lies in an "inaccessible" domain (10). We therefore analyzed in more detail the reactivity of
this position.
Oxalate Protects against Modification of S359C--
Because
positions 355 and 359 are found on the same helical face (Fig.
2B), evidence that position 359 is on the substrate translocation pathway through OxlT could strengthen the idea that this
region forms part of a substrate-binding center. Given the sensitivity
of S359C to MTS-linked probes (Fig. 3), such evidence would be
provided, in part, by demonstration that oxalate (substrate) protects
against inhibition by such thiol-directed agents.
In exploring this possibility, use of MTSCE was preferred over MTSET
for reasons of probe selectivity (see below) and stability (half-lives
of minutes versus hours in aqueous solution at room temperature, respectively (Ref. 17)). Trials to establish a dose-dependence for probe inhibition of the S359C protein showed that a
5-min treatment of proteoliposomes with 10-30 µM MTSCE decreased OxlT function by about 50% (data not given; see Fig. 4A). We also confirmed that
such inactivation took place with an exponential time course (data not
shown) and that the presence of substrate markedly reduced such
inhibition (see below). Since MTSCE had no effect on oxalate transport
by cysteine-less OxlT, parent to S359C, we attributed the action of
MTSCE to a modification of S359C itself. Further, such inhibition
appeared relatively specific to the reaction at position 359 and to the
anionic nature of MTSCE. Partial inhibition of the A358C and I360C
variants required a
These preliminary findings, based on reconstitution of crude extracts,
led us to develop a simple kinetic model as a framework for
interpreting the effects of MTS-linked agents on S359C (see "Experimental Procedures"). To test this model explicitly, we then
moved to work with the purified S359C protein. That work indicated the
reduced activity of S359C relative to its parent stemmed largely from
an increased Michaelis constant (1 mM versus 0.1 mM oxalate), and not a marked change in maximal velocity
(150 versus 220 µmol of oxalate/min/mg of protein,
respectively). In tests of MTSCE inhibition of the reconstituted
material, we found a simple exponential relationship between probe
concentration and the extent of inhibition (Fig. 4A), as
predicted by the kinetic model noted earlier. In a more stringent test,
we chose a probe concentration that yielded near-maximal inhibition,
and then asked how such inhibition was altered in the presence of
oxalate. In that case (Fig. 4B), we recorded a MTSCE and MTSES Approach S359C from Either Surface of
OxlT--
The interpretation of such findings depends importantly on
whether the orientation of OxlT in proteoliposomes is uniformly right
side-out (RSO), as in the intact cell, uniformly inside-out (ISO) or a
mixture of RSO and ISO forms. Because UhpT, a related antiporter, is
found in both RSO and ISO orientations after reconstitution (16), a
mixed distribution would seem the most likely for OxlT. If so, the full
inhibition by external MTSCE and MTSES indicates that the probes gain
access to S359C by moving through pathways present in both
orientations. By contrast, if OxlT orients as either fully RSO or fully
ISO, the approach of such probes to S359C can be confirmed for only one
form of the protein. It was essential, therefore, to establish the
orientation of reconstituted OxlT.
We performed two experiments to address this issue. In one case, we
reconstituted the wild type protein and treated proteoliposomes with
excess trypsin, as previously described in studies with UhpT (16).
Because of the distribution of positively charged residues in
transporters within the major facilitator superfamily (4, 16), we
anticipated that trypsin would cleave at the OxlT cytoplasmic surface,
but not at its extracellular face. Indeed, that oxalate transport was
reduced by nearly 50% (Fig.
5A), in parallel with an
equivalent loss of immunoreactive material (Fig. 5A,
inset), is consistent with this view, provided RSO and ISO
forms have comparable kinetic properties and are present in about equal
proportion, as is true for UhpT (16).
In a second kind of experiment, we monitored the accessibility of
cysteines placed at either end of TM11, using MPB and MTSET, two
water-soluble, thiol-reactive probes of low membrane permeability (16-18).2 As targets, we selected the A345C and A370C
single-cysteine derivatives, because in these variants cysteine is
placed at the cytoplasmic or extracellular border of TM11, respectively
(10)2 (see Fig. 2), and because cysteine at either of these
locations reacts with both maleimide- and MTS-linked probes
(10)2 (Fig. 3). If reconstitution of OxlT yields a
uniformly RSO population, only the A370C derivative should be labeled,
while a fully ISO population would allow labeling of only the A345C
protein. On the other hand, if reconstitution gives a population
containing both RSO and ISO forms, the cysteine on either derivative
will be available to impermeant, external probes. Results obtained in
this kind of experiment clearly favor the last scenario, since both
mutant proteins can be labeled by external MPB (Fig. 5B). Moreover, in each case, MPB modification was blocked by a prior exposure of proteoliposomes to external MTSET, indicating that the
reaction with MPB occurs from the external medium. As well, in this
same experiment, MPB-labeled proteoliposomes were treated with trypsin
to test the idea (Fig. 5A) that only one surface of OxlT
contains an accessible trypsin cleavage site(s). Exposure to trypsin
led to disappearance of the label on A345C, whereas signal derived from
A370C was retained. This verifies the inference that the cytoplasmic,
but not the periplasmic, face of OxlT contains a tryptic cleavage
site(s) and further strengthens the conclusion that MPB labeling is
confined to residues facing the external medium.
Taken together, these two different experimental approaches (Fig. 5)
show that OxlT reconstitutes in mixed orientation, as does UhpT (16),
and that RSO and ISO forms of equivalent kinetic properties are present
in about equal proportion. This conclusion, coupled with the fact that
external MTSCE or MTSES yields full inhibition of S359C (Fig. 4),
shows that these probes are able to approach S359C by entering from
either surface of the protein.
Solute transporters such as OxlT have a network of key residues
whose role is to facilitate substrate movement into and out of an
appropriate binding site, thereby defining a translocation pathway
through the protein (19, 20). One also presumes that, for transporters
of polar molecules such as oxalate, this pathway will be enriched for
residues of a more hydrophilic character than found elsewhere in the
protein (20-24). For these reasons, Lys-355 of OxlT merits close
attention. Not only is Lys-355 the only charged residue in the OxlT
hydrophobic sector (3), it is strategically placed at the center of
TM11 (10), where it might take part in binding of the anionic
substrate, oxalate. If so, one may expect that residues on the same
helical face as Lys-355 might also contribute to the substrate
translocation pathway. Mutagenesis of Lys-355, along with
thiol-specific modification of single-cysteine variants throughout
TM11, has now given strong positive support to these ideas.
Lys-355 Is Essential--
The results of directed mutagenesis
indicate that Lys-355 is critical to OxlT function, for among the
substitutions examined (Lys-355 Assignment of TM11 to the Translocation Pathway--
A cysteine
scan of TM11 strengthens the idea that Lys-355 lies in a region of
significance, since the helical face containing this residue is highly
susceptible to mutational perturbation (Fig. 2). Further work with this
single-cysteine panel led to identification of a variant (S359C) that
is reversibly modified by MTS-linked agents (Fig. 3). Study of S359C
became especially revealing when it was shown that OxlT reconstitutes
as a mixed population (Fig. 5), as does UhpT (16); in both cases, about half the molecules orient as in the intact cell (RSO), and half with
the reverse polarity (ISO) (see Ref. 25 for recent discussion). This
observation is significant, because in oxalate-loaded proteoliposomes, these two orientations should also correspond to the two main conformations in the OxlT catalytic cycle (Fig.
6) (26). Thus, in the absence of external
substrate, efflux of internal oxalate through RSO molecules should
freeze OxlT in the open conformation normally used to receive external
substrate at the beginning of the first half-turnover. By contrast,
efflux through ISO molecules would leave the pathway trapped in the
form used normally to accept internal substrate as the concluding
half-turnover is initiated (Fig. 6). As a result, we are able to ask if
a single residue, S359C, is accessible to a hydrophilic probe (MTSCE,
MTSES) when OxlT adopts the conformation used initiate influx (RSO) as
well as the conformation used to initiate efflux (ISO). This is an important question, since a defining criterion of residues on the
translocation pathway is that within a single catalytic cycle they are
alternately exposed to the cis and trans
environments (via cis- and trans-facing
conformations) (19, 20). That OxlT is fully inhibited by external MTSCE
or MTSES (Fig. 4) indicates that S359C is, in fact, available in both
conformations, fulfilling the requirement needed to assign position 359 to the translocation pathway. Equally significant, the quantitative
analysis of substrate protection suggests that approach of such probes
to their target is blocked by the conformational changes associated
with substrate binding and/or transport (e.g. Fig. 4). This
is taken as strong additional support for the idea that position 359 lies on the pathway taken by substrate itself.
Assignment of S359C to the translocation pathway does not require that
cysteine (in S359C) or serine (in wild type OxlT) take part in
substrate binding, although this may occur. Instead, this interpretation specifies only that position 359 be part of the surface
enclosing substrate as it passes through the protein, across the
membrane (19, 20). Similar arguments were presented when assigning
positions on TM7 to the translocation pathway of UhpT (19, 20), but in
that case the experimental system was complex, requiring comparisons of
probe accessibility in the intact cell and everted membrane vesicles.
We believe the present work is far more convincing in that it exploits
a reconstituted preparation with purified protein under conditions that
allow one to monitor the response of both conformations of the target transporter.
Comparisons with Other Transporters--
UhpT offers instructive
parallels to the work presented here. Thus, in TM11 of UhpT one finds
an intrahelical salt bridge involving Lys-388 and Asp-391
(27). Neither of these charged residues is required for the transport
of glucose 6-phosphate, since normal function is present in mutants
lacking both residues (the K388C/D391S variant) (27). On the other
hand, when TM11 of UhpT contains a lone positive charge (the D391C
mutant), substrate selectivity is biased to favor molecules such
as PEP, which carry an additional negative charge into the active site
(27). At the least, such findings suggest that portions of TM11 lie on the translocation pathway through UhpT and that they can do so without
at the same time directly influencing substrate specificity, much as we
suggest for position 359 in OxlT. Work with UhpT makes it equally
clear, however, that residues lining the pathway may exert a dominant
influence on substrate preference if there is a required electrostatic
interaction between substrate and protein, as we suggest is normal for
Lys-355 in OxlT.
The correspondence between the behavior of OxlT, UhpT, and their
mutants, is in accord with their common membership in the MFS (9). In
this regard, one might also note that within this superfamily of mostly
12-helix transporters, TM11 is the least hydrophobic transmembrane
helix (21). This, considered along with our findings in OxlT and UhpT
(26), leads us to suggest that TM11 will be found to line the transport
pathway in all other examples within the MFS.
Relevance to Mechanism--
It is commonly accepted that, for
energetic reasons, charged residues are infrequently found in
transmembrane
We emphasize that Lys-355 is only one of several residues that may be
involved in construction of the substrate-binding site. Judging from
the OxlT amino acid sequence, and with the assumption that this binding
site lies in the hydrophobic sector (see Refs. 22 and 27), we would
expect TM2 to be of equal importance, since it is the only other OxlT
transmembrane helix with significant polarity. The experimental
determination of OxlT topology2 indicates that, of the 20 residues that can now be assigned to TM2 (Asn-47 through Gln-66), there
are 8 potential hydrogen bond donors (Asn-47, Ser-51, Gln-56, Thr-57,
Thr-60, Ser-62, Gln-63, Gln-66). We suggest, therefore, that binding of
divalent oxalate is accommodated by two distinct mechanisms:
(a) by the electrostatic interaction between one carboxyl
group and Lys-355 on TM11, and (b) by interaction of the
second carboxyl group with the cluster of hydrogen bond donors on
TM2.2 It also seems feasible that, in the absence of
substrate, the net charge on Lys-355 is partly stabilized by the
cluster of electronegative centers (O, N) in TM2. For these reasons, we
consider it significant that TM2 and TM11 are nearest neighbors in the
general structural model for proteins within MFS (21) and in the
specific model generated by biochemical studies of LacY (22).
Cys, Gly, Gln, Arg, or Thr), residual function was found
for only the K355R derivative, in which catalytic efficiency had fallen
2,600-fold. Further insight came from a study of TM11 single-cysteine
mutants, using the impermeant, thiol-specific reagents,
carboxyethyl methanethiosulfonate and ethyltrimethylammonium
methanethiosulfonate. Of the five reactive positions identified in
TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C
and A345C, and G366C and A370C, respectively), whereas the fifth was at
the center of the helix (S359C). Added study with carboxyethyl
methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that
the attack on S359C could be blocked by the presence of the substrate,
oxalate, and that protection could be predicted quantitatively by a
kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such
work contained OxlT of right side-out and inside-out orientations in
about equal amounts. Accordingly, full inhibition of S359C by the
impermeable methanethiosulfonate-linked probes must reflect an approach
from both the cytosolic and periplasmic surfaces of the protein. This,
coupled with the finding of substrate protection, leads us to conclude
that S359C lies on the translocation pathway through OxlT. Since
position 359 and 355 lie on the same helical face, we suggest that
Lys-355 also lies on the translocation pathway, consistent with the
idea that the essential nature of Lys-355 reflects its role in binding
the anionic substrate, oxalate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/formate1
antiporter, OxlT (1-4).
OxlT therefore plays a pivotal role in construction of a "virtual"
proton pump, an organizational scheme
with relevance to several aspects of microbial cell biology (4-6).
Biochemical study (2, 7, 8) suggests that OxlT, a member of the major
facilitator superfamily (MFS) (9), may also serve as a valuable model
for more broadly directed studies of membrane transport proteins. For
such reasons, further study of OxlT may contribute to ongoing studies
of both the biology and biochemistry of membrane transport.
-helices, a
characteristic shared by most other members of the major facilitator
superfamily (9). This analysis also predicts that Lys-355 is positioned
near the center of transmembrane helix 11 (TM11)1 (3), an expectation
verified by site-directed fluorescent labeling of
single-cysteine variants in this vicinity (10). Direct evaluation of
OxlT topology2 suggests that
Lys-355 does not interact with a nearby anionic residue, thereby
raising an apparent paradox. Placement of an uncompensated charge
within a hydrophobic environment adds a destabilizing element to
membrane protein structure (12, 13), but OxlT can be remarkably stable
in lipid-detergent micelles (7, 10). This contradiction might be
reconciled if the energetic disadvantage contributed by Lys-355 were
used to offset the energetic cost of binding and/or transporting
substrate anions. Such compensating effects could be achieved in a
simple way if TM11 forms part of the substrate translocation pathway,
allowing direct interactions between positively charged Lys-355 and
negatively charged substrate. That solubilized OxlT is intensely
stabilized by the presence of its substrate anions (7, 10), gives
indirect support to this view, but more concrete observations are
necessary if this model is to guide further work.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside to
cells in the mid-exponential phase of growth; cells were harvested after an additional 4 h growth.
-mercaptoethanol (1). Solubilized protein (0.7-1
mg/ml) was reconstituted by detergent dilution (10, 15) to give
proteoliposomes loaded with 100 mM potassium oxalate, 50 mM MOPS/K (pH 7).
-mercaptoethanol, or by four cycles of washing with 20 mM potassium phosphate (pH 7), as noted. Subsequently,
membrane protein was solubilized and reconstituted as described above. These conditions allow labeling of residues on both intra- and extracellular surfaces2 (10).
95%) was achieved by raising probe
concentration to 300 µM. However, even at such high
levels of MTSCE, sufficiently high concentrations of oxalate afforded
complete protection. Because 2 mM MTSCE had no effect on
oxalate transport by the cysteine-less parent (not shown), inhibition
of the S359C derivative was attributed solely to reaction of the probe
with the target cysteine at position 359, justifying use of a simple
kinetic scheme to model probe inhibition. We assumed unliganded OxlT
(C) could interact with either substrate (S) or the probe (P),
generating, respectively, either liganded OxlT (CS) or an irreversibly
inhibited complex (CP*).
or
(Eq. 1)
S and P represent substrate (oxalate) and probe concentrations,
respectively, t is time, k is the rate constant
governing probe modification (Equation 2), and
Kd is the dissociation constant for the liganded
complex, CS. This model was used to evaluate probe interactions at
Ser-359, using material purified as described above for the A345C and
A370C proteins.
(Eq. 2)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Lys-355 is essential to normal OxlT function.
A, proteoliposomes were prepared from extracts of
wild type OxlT and from mutants in which Lys-355 was replaced by Arg,
Thr, and Gln. (The K355C mutant is described below.) Oxalate-loaded
proteoliposomes suspended in Buffer B were given 1 mM
labeled substrate. Samples were taken at the indicated times for
filtration and washing to determine [14C]oxalate
accumulation. B, cartoon showing the organization of TM 11;
amino acids are given using the single letter code.
5% the parental level) for this analysis, but all others were present at levels high enough (
40% normal) (10) to assess functional status. Among the 27 expressed proteins, two (G349C and K355C) gave undetectable levels of
[14C]oxalate transport; three others (A354C, G362C,
G363C) gave marginal responses (0.2-1% residual activity). In all
other cases, we found activity corresponding to at least 1% of the
cysteine-less parental protein, yielding a signal-to-noise ratio of at
least 20. Since the turnover number for OxlT is at least 1000-fold
greater than usually observed for membrane transporters (1, 4, 8), even
these low relative levels of activity might reflect significant rates
of anion exchange. Such findings, including the null behavior of K355C,
are consistent with earlier studies (10) and with the results noted
above (Fig. 1).
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Fig. 2.
Oxalate transport by TM11 single-cysteine
mutants. Left, OxlT function was evaluated for a
single-cysteine panel spanning TM11 (S344C-F373C). Initial rates of
[14C]oxalate transport, normalized for known levels of
OxlT expression (10), are given relative to that of the cysteine-less
parental protein, whose activity was 4.5 µmol/min/mg of protein
(horizontal line). In three cases
(asterisks), lack of OxlT function was due to lack of
expression. Except for S359C, data reflect mean values ± S.D.
found in three independent trials; for S359C, data from nine separate
experiments are included. Brackets on the
horizontal axis indicate the TM11 core domain
(10). Right, helical wheel representation of the initial
rate data for the 11 residues in the TM11 core domain (F351C to F361C).
Relative activities (from A) are displayed on the
perimeter.
10% parental) are broadly represented throughout TM11, a
striking distribution is found in this inaccessible region. Within this core, cysteine substitutions yielding low specific activity are restricted to the helical face containing Lys-355 (Fig. 2B),
indicating a distinct functional asymmetry in this area.
).
-mercaptoethanol. Subsequent to this in situ labeling, protein was solubilized, and assays of transport by
proteoliposomes recorded residual function. MTSET and/or MTSCE gave
significant inhibition at five positions, two each in the TM11
cytoplasmic (S344C, A345C) and periplasmic (G366C, A370C) domains (Fig.
3, top), and one (S359C)
within the TM11 core region (Fig. 3, bottom). In each of
these cases, there was unambiguous reversal of inhibition by
-mercaptoethanol, suggesting an unrestricted access to the target
cysteines by the probes (despite their differing charge), and an
equally free access to their modification products by the
mercaptoethanol. In two instances MTSCE, but not MTSET, gave an
irreversible block (A354C, A358C), but the partial nature of this
inhibition (
40%) did not suggest further study would be helpful.
Finally, we noted that I360C, also within the TM11 core, responded to
MTSET with prominent inhibition but incomplete reversibility, and to
MTSCE with a partial inhibition that was similarly not reversed.
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Fig. 3.
Effects of thiol-specific probes on OxlT TM11
single-cysteine variants. Membrane ghosts of the indicated OxlT
single-cysteine derivatives were incubated for 1 h at 23 °C in
20 mM potassium phosphate (pH 8) with either 1 mM MTSET or 2 mM MTSCE. To quench the reaction,
membranes were centrifuged and washed four times using 20 mM potassium phosphate (pH 7), with or without 5 mM -mercaptoethanol, as indicated. Subsequently, protein
was solubilized and reconstituted, and initial rates of
[14C]oxalate transport were determined (see Fig. 2). For
each derivative, rates are expressed relative to those of control
preparations not exposed to probes. In most cases, data reflect mean
values (± S.D.) from three independent experiments; for derivatives
showing
50% inhibition, a fourth experiment was performed.
Upper panel, cysteines in positions classified by earlier
work (10) as exposed to cytoplasmic (positions 344-349) and
periplasmic (positions 362-373) surfaces. Lower panel,
cysteines at positions identified (10) as the TM11 core (positions
351-360) (see Fig. 2). For clarity, arrows are used to
indicate variants significantly (>50%) affected by MTSET or
MTSCE.
5-min treatment with 1-2 mM MTSCE
(not shown; see Fig. 3), and while MTSET inhibition of S359C
(e.g. Fig. 2) was found, comparable effects required
5-10-fold greater concentrations of MTSET than MTSCE (data not
given).
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Fig. 4.
Substrate protects S359C against MTSCE.
A, dose dependence. After reconstitution of the purified
S359C protein, oxalate-loaded proteoliposomes trapped on a Millipore
filter were overlaid for 5 min with 0.4 ml of Buffer B containing MTSCE
as indicated. Residual fractional activity (F) was measured
by a second overlay using Buffer B with 100 µM labeled
substrate. B, substrate protection. As in A,
proteoliposomes were incubated with 300 µM MTSCE, with
and without potassium oxalate of the designated concentration. Residual
activity (F) is given as a function of oxalate present in
the preincubation period. C, evaluation of
KD. Using data from B, a plot of
( 1/lnF) versus oxalate concentration was
constructed. A calculated value for the OxlT Kd (2.4 mM) was derived using the model developed under
"Experimental Procedures."
95%
inhibition by 300 µM MTSCE alone, with progressively
diminishing effects as oxalate was introduced. At 10-20 mM
oxalate, the MTSCE block was reduced by half, and at 100 mM
oxalate there was a nearly complete rescue. More important, throughout
this range, the degree of protection was predictable by the model,
given an OxlT dissociation constant (Kd) of 2.4 mM (Fig. 4C). In four other trials of similar
design, using either purified protein or crude extracts, we calculated
Kd values of 1.5-3.3 mM (mean ± S.E. of 2.2 ± 0.4 mM) for both MTSCE (at 30 or 300 µM) and MTSES (at 300 µM). This derived
Kd value is compatible with the elevated Michaelis
constant for oxalate exchange by the S359C mutant (see above), and is
also consistent with an independent estimate of Kd
(2.4 mM), based on use of oxalate to stabilize against
thermal denaturation (as described in Refs. 7 and 8) (data not shown).
Such findings (Fig. 4) confirm the accessibility of S359C to
MTS-linked probes and document that this behavior is described by a
simple scheme that assumes access to position 359 is possible only when
OxlT is in an unliganded state (see "Experimental Procedures"). We
also note that inhibition by MTSCE or MTSES must reflect probe action
from the external medium, because an attack from the inner surface (e.g. following any slow inward diffusion of probe) would
not take place in the presence of 100 mM internal oxalate.
Further, because full inhibition occurs in the absence of substrate,
external probe must have access to the entire OxlT population.
View larger version (35K):
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Fig. 5.
Mixed orientation of OxlT in
proteoliposomes. A, trypsin susceptibility. After
reconstitution of purified wild-type protein, proteoliposomes were
placed in Buffer B with ( ) or without (
) 1 mg/ml trypsin. At the
indicated times, the reaction was quenched by 25-fold dilution into
iced Buffer B containing 0.5 mM PMSF. After centrifugation
and resuspension in Buffer B, residual OxlT function was determined by
the traditional filtration assay, using 60-s incubations with 100 µM labeled substrate. Inset, a Western blot
showing OxlT present after the 60-min incubation in the presence (+) or
absence (
) of trypsin, using a polyclonal antibody reactivity with
the OxlT N terminus (3). B, thiol labeling of the ends of
TM11. The A345C and A370C proteins were separately purified and
reconstituted, after which thiol labeling and trypsin treatment were
performed as noted in A and under "Experimental
Procedures." Upper panel, detection of MPB labeling. For
each protein, proteoliposomes were divided into four portions of 0.5 ml
each, corresponding to the lanes shown: a, solvent control;
b, MPB treatment; c, MPB treatment after exposure
to MTSET; d, MPB treatment followed by trypsin treatment.
Lower panel, detection of OxlT content. To control for
recovery of OxlT, the blot used to record MPB labeling
(upper panel) was stripped and re-probed with the
N-terminal polyclonal antibody. For the two samples treated with
trypsin, we assume part of the diminished signals reflect cleavage of
the ISO forms present in both A345C and A370C populations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Cys, Gly, Gln, Arg, or Thr), only
the K355R variant retains any vestige of function (Fig. 1). In that
case, however, catalytic efficiency is markedly reduced, reflecting
reductions in both the affinity for and velocity of oxalate transport.
The phenotype of such mutants leads us to conclude that anion transport by OxlT requires a positive charge, preferably lysine, at position 355. The kinetic changes found in the K355R mutant also provide direct
evidence supporting the proposed role of Lys-355 in substrate binding.
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Fig. 6.
Accessibility of S359C in reconstituted
OxlT. The drawing depicts the mixed orientation of OxlT found in
proteoliposomes prepared by detergent dilution. The high concentration
of internal substrate (here, 100 mM oxalate) drives efflux
of substrate during a single half-turnover for both RSO and ISO
orientations. Lack of external substrate prevents completion of the
catalytic cycle, leaving the translocation pathway in both kinds of
molecules exposed to the external medium. The diagram also shows the
relative positions of S359C and Lys-355 for RSO and ISO
orientations.
-helices, an expectation generally borne out in
surveys of transmembrane helices known at atomic resolution (see Ref.
10). The same appears true if less direct criteria are used, as in UhpT
or LacY, where one finds that such charged residues may be organized by
intra- or interhelical salt bridges (11, 26, 28, 29). Accordingly, the
presence of uncharged residues in the hydrophobic sector of such
membrane proteins is viewed as introducing inappropriate destabilizing
influences. For proteins involved in electron transport, where a fixed
geometry between prosthetic groups may be of paramount importance, this
argument would appear justified. However, just this kind of structural
flexibility could be of advantage to solute carriers and facilitators,
which adopt multiple conformations as they enclose and transport their
substrates. Indeed, demonstration that TM11 lies on the OxlT transport
pathway leads us to suggest that such cost-benefit considerations may
be essential to this antiporter. Thus, we imagine the oxalate-binding
site comprising liganding groups arising from several transmembrane
helices. One of these groups is Lys-355, whose presence should, for
thermodynamic reasons, promote a conformation that is "open" to the
external hydrophilic environment and at the same time establish the
electrostatic driving force that attracts negatively charged substrate,
oxalate. Once within the transporter, the formation of an ion pair
between Lys-355 and a substrate carboxyl would enable closure of the
translocation pathway, which may then reopen spontaneously to face
either membrane surface. If substrate dissociates when the pathway
faces the original (cis) surface, no net substrate movement
occurs; but if the pathway is facing the opposite (trans)
surface when substrate leaves, net transport will occur. This
mechanistic view is not only suggested by our findings, it may also
rationalize the elevated Kd and
Km found for the S359C variant. Thus, the proximity of Lys-355 may lower the pKa of the thiol at
position 359, placing the anionic sulfide group (-S
) in
the pathway and reducing fractional occupancy of the pathway by the
anionic substrate. Placement of a deprotonatable group in the pathway
may also have consequences for the electrogenic character of the
heterologous oxalate:formate exchange, a question to be explored in
later work.
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FOOTNOTES |
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* This work was supported by Research Grants MCB-9603997 and MCB-9986617 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Biology Department, Brookhaven National
Laboratory, Upton, NY 11973.
The authors contributed equally to this work.
§ Present address: Dept. of Molecular and Cell Biology, Graduate School of Agricultural Sciences, Tohoku University, Sendai 981-8555, Japan.
¶ To whom correspondence should be addressed. Tel.: 410-955-8325; Fax: 410-955-4438; E-mail: pmaloney@jhmi.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008417200
2 Ye, L., Jia, Z., Jung, T., and Maloney, P. C. (2001) J. Bacteriol., in press.
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ABBREVIATIONS |
---|
The abbreviations used are:
TM, transmembrane;
MTSCE, carboxyethyl methanethiosulfonate;
MTSET, ethyltrimethylammonium
methanethiosulfonate;
MTSES, ethylsulfonate methylthiosulfonate;
MPB, 3-(N-maleimidylpropionyl)biocytin;
octylglucoside, octyl--D-glucopyranoside;
AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride;
PMSF, phenylmethylsulfonyl
fluoride;
ISO, inside-out;
RSO, right side-out;
MOPS, 4-morpholinepropanesulfonic acid;
MFS, major facilitator
superfamily.
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