| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 50, 39130-39136, December 15, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
andFrom the Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands
Received for publication, July 6, 2000, and in revised form, September 15, 2000
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The citrate transporter of Leuconostoc
mesenteroides (CitP) catalyzes exchange of divalent anionic
citrate from the medium for monovalent anionic lactate, which is an end
product of citrate degradation. The exchange generates a membrane
potential and thus metabolic energy for the cell. The mechanism by
which CitP transports both a divalent and a monovalent substrate was
the subject of this investigation. Previous studies indicated that CitP
is specific for substrates containing a 2-hydroxycarboxylate motif,
HO-CR2-COO In recent years a growing number of secondary transporters have
been discovered that generate rather than consume metabolic energy.
These transporters have been termed precursor-product exchangers since
they catalyze the uptake of a substrate into the cell coupled to the
exit of a metabolic end product into the medium (for a review see Ref.
1). An example of such a transporter is the citrate transporter (CitP)
found in Leuconostoc mesenteroides (2, 3), which catalyzes
the uptake of divalent citrate into the cell coupled to the exit of
monovalent lactate, a metabolic end product of citrate degradation in
lactic acid bacteria (4). The net charge movement over the membrane
during the exchange results in a membrane potential of physiological
polarity. This, in combination with the consumption of a cytoplasmic
proton in the breakdown of citrate, results in a proton motive force
and thus generates metabolic energy for the cell. Recovery from acidic stress and resistance against lactate toxicity have recently been suggested as alternative roles for the metabolic pathway (5).
The ability of CitP to transport two such different substrates as
citrate and lactate is associated with a high specificity for the
2-hydroxycarboxylate motif present in both substrates (i.e.
HO-CR2-COO CitP belongs to the 2-hydroxycarboxylate transporter (2-HCT) family (7)
that contains members found in several lactic acid bacteria as well as
in Bacillus subtilis and Klebsiella pneumoniae (8). The family contains precursor-product exchangers like CitP and the
malate transporter MleP of Lactococcus lactis, but also the Na+/citrate symporter CitS of K. pneumoniae, suggesting that precursor-product exchangers are
"normal" secondary transporters that have been optimized to
catalyze exchange. Transporters of the 2-HCT family are believed to
consist of 11 transmembrane segments (TMSs), based on topology studies
of CitS of K. pneumoniae (9-11). The C terminus of the
proteins resides in the periplasm. A recent study of chimeras between
the citrate transporter CitP and the malate transporter MleP indicated
that the C-terminal region including TMS XI forms part of the
substrate-binding site that interacts with the R groups of
the substrates (8). The C-terminal region contains two conserved arginine residues, one of which is located in TMS XI. The latter residue was thought to be a good candidate for an interaction with the
second carboxylate of divalent substrates.
In the present study Arg-425 in TMS XI of CitP was replaced by
lysine and cysteine residues. The mutant transporters were analyzed for
their affinity toward the divalent and monovalent substrates
(S)-malate and 2-HIB, respectively. In addition, the effect
of chemical modification of residues at position 425 and the ability of
substrate to protect against modification was investigated. It is
concluded that Arg-425 is located in the substrate-binding site where
it is responsible for a high affinity interaction with the second
carboxylate of di- and tricarboxylates.
Bacterial Strains and Growth Conditions--
L. lactis
strain NZ9000 is an MG1363 derivative
(pepN::nisRnisK; (12)) that transports
neither citrate nor malate. The nisR and
nisK genes were inserted in the chromosome to allow induced expression of plasmid-encoded genes under control of the tightly regulated nisA promoter (12). Cells harboring the expression vector pNZcitP containing the citP gene (see below) were
grown at 30 °C in closed serum bottles and without shaking in M17
broth (Difco) supplemented with 0.5% (w/v) glucose and 5 µg/ml
chloramphenicol. Unless otherwise stated, expression of the transporter
was induced by growing the cells to an optical density of 0.6 measured
at 660 nm (OD660), followed by addition of a 1000-fold
dilution of the supernatant of an overnight culture of the nisin
producing L. lactis strain NZ9700 into the cultures (14).
The supernatant contained approximately 10 ng of nisinA/ml (15). Growth
was continued for 1 h followed by harvesting of the cells by
centrifugation. In some cases a lower level of induction was required
to allow kinetic analysis (see "Results"). Then, cells expressing
CitP were grown in the presence of a 30,000-fold dilution of the nisin containing supernatant for 0.5 h.
DNA Manipulations--
General procedures for cloning and DNA
manipulations were performed essentially as described by Sambrook
et al. (16). Expression vector pNZcitP codes for the CitP
protein with 10 additional histidine residues at the N terminus (8).
R425C and R425K mutations were introduced in the citP gene
in two steps by overlap extension polymerase chain reaction (17). All
polymerase chain reaction-amplified DNA fragments were sequenced to
confirm the nucleotide sequence. Ligation mixtures were transformed to
L. lactis NZ9000 by electroporation as described by Holo and
Nes (18).
Preparation of Right-Side-Out Membrane
Vesicles--
Right-side-out (RSO) membrane vesicles of NZ9000 cells
expressing wild type or mutant CitP were prepared by the osmotic shock lysis procedure in the presence of 5 mM
(S)-malate, essentially as described previously (6).
Membrane vesicles were washed once with 50 mM
potassium phosphate, pH 6, containing 5 mM
(S)-malate and concentrated by centrifugation in an
Eppendorf table top centrifuge operated at full speed for 5 min. The
internal pool of (S)-malate was labeled with
(S)-[14C]malate by incubating the concentrated
membranes with 186.7 µM L-[1,4(2,
3)14C]malate for 1 h in the presence of 1 mM valinomycin and 0.5 mM nigericin. Protein
concentrations were determined as described by Lowry et al.
(19). In case the vesicles were used for chemical modification studies,
(S)-malate was omitted in the preparation procedure, and
loading of the vesicles with (S)-[14C]malate
was done as described under "Chemical Modification."
Exchange Measurements--
Aliquots of 2 µl of
(S)-[14C]malate-loaded vesicles were diluted
into 200 µl of 50 mM potassium phosphate, pH 6, containing substrates at the indicated concentrations at 20 °C.
Internal radioactivity was determined at different time points by
dilution with ice-cold LiCl followed by rapid filtration, as described (6). Final membrane protein concentrations in the assays were between
250 and 350 µg/ml. To evaluate the data, initial rates of exchange
were determined by fitting the data to an exponential decay using
nonlinear fitting procedures provided by the Sigma Plot software
(Jandel Scientific, San Rafael, CA), as described previously (7). The
rate of efflux in the absence of external substrate was subtracted from
the observed rates. Exchange rates were determined at different
external substrate concentrations to estimate the affinity constant
Kmapp. The data were fitted to an
equation describing competitive inhibition, taking into account the
effect of (S)-[14C]malate in the external
buffer caused by the dilution of the (S)-[14C]malate-loaded membrane vesicles
(6).
Chemical Modification--
RSO membrane vesicles (10 mg
protein/ml) prepared in the absence of (S)-malate were
incubated for the indicated times in 50 mM potassium
phosphate containing 5 mM of the lysine-specific reagent
TNBS (Fluca) or 1 mM of the sulfhydryl reagents MTSEA, MTSES, MTSET (Anatrace), pCMB, and pCMBS (Sigma) at 23 °C. Reactions with the sulfhydryl reagents were performed at pH 6 and with TNBS at a
range of pH values. Following incubation, the vesicles were diluted 15 times in 50 mM potassium phosphate, pH 6, and concentrated by centrifugation in an Eppendorf table top centrifuge operated at full
speed for 5 min. The membranes were washed 3 times using the same
procedure. To load the vesicles with
(S)-[14C]malate, the concentrated vesicles
were incubated overnight at 4 °C in the presence of 5 mM
(S)-malate, 186.7 µM
L-[1,4(2,3)14C]malate, 1 mM
valinomycin, and 0.5 mM nigericin. Exchange activity was
determined as described above.
To test the effect of substrate on chemical modification, RSO membranes
were preincubated for 2 h with and without the substrate in the
presence of 1 mM valinomycin and 0.5 mM
nigericin followed by reaction with 0.1 mM pCMB for 0.5 min
or with 30 mM TNBS for 2 h at pH 7. The membranes were
washed twice with 50 mM potassium phosphate, pH 6, in the
presence of substrate and 4 times in the absence of substrate. The
concentrated vesicles were loaded with 5 mM radiolabeled
(S)-malate, and exchange activity was determined as
described above. To evaluate the efficiency of the washing procedure
for removing internal substrate, vesicles were equilibrated with 60 mM 2-HIB, butyrate, (S)-malate,
(S)-citramalate, or succinate and were washed four times. In
all cases exchange rates were not significantly different from vesicles
that were incubated in the absence of substrate (data not shown).
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot
Analysis--
Right-side-out membrane vesicles were subjected to
SDS-polyacrylamide gel electrophoresis using a 12% polyacrylamide gel
matrix (15 µg protein/lane). After electrophoresis, the proteins were transferred to poly(vinylidene difluoride) membranes and analyzed using
monoclonal antibodies directed against a His tag (Dianova, Hamburg,
Germany). Antibodies were visualized using the Western light
chemiluminescence detection kit (Tropix, Bedford, MA).
Chemicals--
L-[1,4(2,3)-14C]Malic
acid (51 mCi/mmol) was obtained from Amersham Pharmacia Biotech. All
other compounds were obtained from Fluka (Buchs, Switzerland) or Sigma.
Construction and Activity of the R425C and R425K Mutant
Transporters--
Arg-425 is conserved in the transporters of the
2-HCT family and located in the C-terminal putative TMS XI (Fig.
1). The involvement of Arg-425 in the
interaction with the second carboxylate present in divalent substrates
was investigated by constructing site-directed mutants of the citrate
transporter CitP of L. mesenteroides. Arg-425 was replaced
with Lys, a conservative mutation that retains the positive charge, and
with the neutral Cys residue. Mutant and wild type transporters were
N-terminally tagged with 10 histidines and expressed in L. lactis NZ9000 cells using the inducible nisA promoter system. The expression levels in the membrane were
analyzed by immunoblotting using antibodies directed against the His
tag. The apparent molecular mass of the proteins was about 40 kDa, and
under the same induction conditions, the expression levels were similar
for CitP and both mutants (Fig. 2).
Exchange provides a sensitive assay for the activity of CitP (7).
Mutant transporters were assayed for homologous exchange in
right-side-out membrane vesicles, using the high affinity substrate (S)-malate. Vesicles loaded with 5 mM
radiolabeled (S)-malate were diluted 100-fold into buffer.
In the absence of external substrate, efflux of (S)-malate
from the membranes down the concentration gradient was a slow process
for both wild type and mutant transporters (Fig.
3 ( Affinity for (S)-Malate and 2-HIB--
The kinetic characteristics
of the mutant transporters were analyzed with 2-HIB and
(S)-malate, a mono- and a divalent substrate of CitP,
respectively (see Fig. 4). Under standard
induction conditions, exchange catalyzed by wild type CitP was so fast
that initial rates of transport could not be measured (Fig.
3A). To allow the analysis, CitP expression levels were
reduced by varying the inducer concentration in the growth medium (see
"Experimental Procdures").
The Kmapp for external
(S)-malate at an internal concentration of 5 mM
(S)-malate was 90 µM for wild type CitP in
fair agreement with previous reports (6, 8). In heterologous exchange
using the same concentration of internal (S)-malate, the
Kmapp for external 2-HIB was 5 mM (Fig. 5A and
Table I). Replacing Arg-425 by Lys, thus
conserving the positive charge at position 425, reduced the affinity
for (S)-malate about 10-fold (Fig. 5B and Table
I). Substitution for the neutral Cys residue had a much more dramatic
effect on affinity. No saturation was observed up to a concentration of
10 mM (Fig. 5C). In contrast to the decrease in
the affinity for divalent (S)-malate, the R425K and R425C
mutants revealed an increased apparent affinity for monovalent 2-HIB. The affinity of R425K increased 4-fold, whereas R425C had a 20-fold higher affinity for 2-HIB compared with wild type CitP. The maximal rates of exchange decreased in the order CitP > R425K > R425C for both (S)-malate and 2-HIB (Fig. 5 and Table
I).
The results suggest that Arg-425 is specifically involved in the high
affinity toward the divalent (S)-malate. The positive charge
of the Arg seems to play an important role in the interactions since
the changes in (S)-malate affinity were much less pronounced when Arg-425 was replaced with the positively charged Lys residue.
Reactivation of the R425C Mutant--
The methanethiosulfonate
(MTS) derivatives MTSEA, MTSES, and MTSET are small, charged,
water-soluble, and cysteine-specific reagents (20). They form a mixed
disulfide with the thiol of the cysteine via the addition of
-SCH2CH2X groups where X
is SO3 Chemical Modification of R425K by TNBS--
TNBS reacts
specifically with lysine residues and/or the N terminus of proteins
(22, 23) introducing a covalently linked trinitrophenyl group in the
protein. Increased reactivity at higher pH values is characteristic for
a specific reaction with an amino group (23). Treatment of
right-side-out membrane vesicles containing R425K with 5 mM
TNBS at pH 6 did not reduce the exchange activity of the transporter.
However, the same treatment at pH values of 7 and 8 resulted in the
loss of 35 and 80% exchange activity, respectively (Fig.
7A). Under the same reaction
conditions, TNBS had no effect on wild type CitP (Fig. 7B)
indicating a specific modification of the lysine at position 425 in the
R425K mutant. Apparently, modification of Lys-425 in the R425K mutant
renders an inactive transporter.
Accessibility of Position 425--
The localization of the Cys
residue at position 425 in the R425C mutant with respect to the
membrane was investigated by the reactivity with membrane-permeable and
-impermeable thiol reagents. MTSET is a strong base and MTSES a strong
acid, and both are generally regarded to be poorly membrane-permeable.
On the other hand, MTSEA is a weak base that equilibrates across the
membrane in its undissociated form (24). Chemical modification of the
R425C mutant by membrane-impermeable MTSES and MTSET was incomplete
even after long incubation times (Table II). The incomplete
inactivation was due to low inactivation rates as higher reagent
concentrations increased inactivation (not shown). In contrast, the
time course of activation with membrane-permeable MTSEA was much
shorter. The stimulatory effect was saturated within 10 min.
pCMB and its sulfonic acid derivative pCMBS are organomercurial
reagents that react with high specificity with cysteine residues. pCMB
is membrane-permeable, and pCMBS is not. Treatment of right-side-out membrane vesicles containing R425C with either pCMB or pCMBS eventually resulted in complete inhibition of homologous (S)-malate
exchange (Table II). The same treatment had no effect on exchange
catalyzed by the wild type transporter, indicating that the Cys at
position 425 was modified. Inactivation of the R425C mutant by pCMBS
was much slower than observed for of pCMB which fully inactivated the
transporter within 3 min (Table II). In fact, 0.5 min of
incubation with 0.1 mM pCMB resulted in 80% inhibition
(see below), while the same level of inhibition by pCMBS required 10 min of incubation at a 10-fold higher concentration. Taken together
these results indicate that Cys-425 reacts much faster with
membrane-permeable reagents, suggesting that the residue is accessible
at the cytoplasmic side of the membrane.
Substrate Protection against Chemical Modification--
The R425C
mutant revealed a relatively high affinity for external 2-HIB in
heterologous exchange (Kmapp = 0.22 mM; Table II) and, therefore, this substrate was
selected to see whether the presence of substrate could protect the
mutant against inactivation by pCMB. Right-side-out membrane vesicles were incubated with 2-HIB to equilibrate the substrate over the membrane, followed by treatment with pCMB, and subsequent removal of
the substrate and unreacted pCMB (see "Experimental Procedures"). In the absence of 2-HIB, the treatment resulted in 80% inhibition of
exchange activity (Fig. 8A).
2-HIB protected R425C against pCMB inactivation in a
concentration-dependent manner, but much higher
concentrations were required than anticipated. Protection of R425C by
2-HIB was incomplete even at 60 mM which is far above the
Kmapp for external 2-HIB. In the
control experiment, 2-HIB was replaced by butyrate, which is very
similar to 2-HIB but lacks the hydroxyl group and therefore is not a
substrate of CitP (7). The presence of butyrate did not affect the
inactivation by pCMB (Fig. 8A), indicating that the
protection by 2-HIB is specific.
(S)-Malate and another high affinity substrate of CitP
(S)-citramalate (6) were tested for their potency to protect
the R425K mutant against inactivation by TNBS (Fig. 8B). In
the presence of 60 mM (S)-malate, the 70%
inhibition observed in the absence of substrate was reduced to 30%,
whereas 60 mM (S)-citramalate completely
protected against inactivation under the conditions of the experiment.
Similarly as observed for 2-HIB above, much higher concentrations of
the substrates were required than would be expected based on the
affinity constants for external (S)-malate in the homologous
exchange reaction. In the control experiment, succinate which lacks the
2-hydroxy group of (S)-malate, and therefore is not a
substrate of CitP, had no effect on the inhibition by TNBS.
In this study, the mechanism by which CitP transports both
divalent and monovalent substrates, which is crucial to the
physiological function of membrane potential generation by the
transporter, was investigated. Previous substrate specificity studies
showed that CitP is specific for substrates containing a
2-hydroxycarboxylate motif, HO-CR2-COO It is concluded that Arg-425 indeed interacts with the second
carboxylate of the divalent substrates, based on the following observations. First, mutation of Arg-425 decreased the affinity for
divalent (S)-malate but not for monovalent 2-HIB. Second, Arg-425 is not essential for transporter activity. Third, a positive charge at position 425 resulted in improved transport activity. Fourth,
chemical modification of the residue at position 425 significantly affected transporter activity. Finally, the presence of substrate protected the residue at position 425 against chemical modification. The combination of the results strongly suggests that Arg-425 is in the
substrate-binding pocket where it interacts with the second
carboxylate. The involvement of TMS XI in the binding site is in line
with the behavior of chimeric transporters (8).
The affinities of the mutant and wild type transporters were determined
by homologous and heterologous exchange using membrane vesicles loaded
with a fixed (S)-malate concentration and varying the
external substrate concentrations. The determined affinities are for
the external substrate. In a ping-pong type exchange mechanism, the
apparent affinity for the external substrate is dependent on the
affinity and concentration of the internal substrate. Non-saturating conditions at the inside result in higher apparent affinities for the
external substrate and lower maximal rates. Consequently, a mutation
that lowers the affinity for internal (S)-malate decreases the Kmapp for external substrate
and the maximal rate of exchange. The kinetics observed with 2-HIB of
the R425K and R425C mutants are consistent with a decrease in internal
(S)-malate affinity in the order Arg-425, Lys-425, Cys-425,
if the binding affinity for 2-HIB would not change significantly. The
kinetic affinity for external 2-HIB increases, and the maximal rate for
heterologous 2-HIB/(S)-malate, as well as for homologous
(S)-malate exchange, decreases (Fig. 5). The observed
decreased affinity of the mutants for external (S)-malate in
homologous exchange strongly argues in favor of a decreased binding
affinity of the protein for (S)-malate with a subsequent
lowering of the kinetic affinity for internal (S)-malate.
The guanidinium group of the Arg-425 residue is not absolutely
essential for transport function since even a non-conservative substitution for Cys does not abolish the activity of CitP. However, the affinity for divalent (S)-malate was severely affected
by this mutation. The drop in affinity was less when Arg-425 was mutated to Lys, and, especially, the reactivation of R425C by MTSEA,
which restores a positive charge at position 425, strongly suggests
that the positive charge is essential for high affinity binding of
divalent substrates. Nevertheless, the positive charge is not the only
relevant factor since Arg-425 cannot be replaced with lysine or
MTSEA-labeled cysteine without some loss of affinity. The chemistry of
the guanidinium group allows for the formation of two hydrogen bonds,
which is known to result in an interaction of unusual strength with a
carboxylate group (25). Alternatively, only an Arg residue can position
its charge accurately relative to the substrate for an optimal
interaction. In contrast to the stimulation of exchange by MTSEA,
positively charged MTSET inhibited the R425C mutant. The three methyl
groups of the quaternary ammonium group of the reagent are likely to
interfere sterically with substrate binding and thus inactivate the
transporter. MTSES, pCMB, and pCMBS inactivated R425C and TNBS R425K
most likely because of the presence of a negative charge on the side
chains that is expected to repel the carboxylate of the substrate
and/or by steric interference with the substrate.
In the topology model of CitP, Arg-425 is located at the cytoplasmic
side of TMS XI (9-11). The present data support this localization. The
chemical inactivation of the R425C mutant in right-side-out vesicles
was much faster for the membrane-permeable pCMB than for its
impermeable counterpart pCMBS (Table II). Since the two probes have
similar chemical reactivities, the difference in inactivation rate
suggests that the reactive cysteine is only accessible from the inside.
Similarly, chemical modification by the membrane-impermeable MTSET and
MTSES was very slow, and modification by the permeable MTSEA was fast.
These low reaction rates are not due to differences in chemical
reactivity with the thiol groups. In fact, MTSET is known to have
2.5-fold higher intrinsic reactivity for sulfhydryls than MTSEA (13).
Thus, these results also suggest that the reaction takes place at the
internal face of the membrane. The low rate of modification with pCMBS,
MTSES, and MTSET may be explained by a slow permeation through the
membrane by these reagents followed by reaction at the cytoplasmic side
or by a low accessibility of the Cys residue from the outside.
Accessibility of a binding site residue from either side of the
membrane is not unlikely since the transporter has to expose the
binding site alternately to the two sides of the membrane during turnover.
The presence of substrate protected against chemical modification of
the residue at position 425, which is consistent with the assignment of
Arg-425 as a binding site residue. Interestingly, 2-HIB protected R425C
against pCMB inactivation in a concentration-dependent manner, but protection was still incomplete at a concentration of 60 mM, indicating a very low affinity for the substrate. This contrasted with the high apparent affinity for external 2-HIB in the
exchange assays, Kmapp = 0.22 mM (Table I). The same situation was observed for the protection of R425K by (S)-malate against inhibition by
TNBS. Possible explanations for the apparent discrepancy may be the underestimation of the binding affinity for external substrate when
deduced from a kinetic experiment as discussed above or, alternatively,
the transporter has a much lower affinity for internal substrate than
for external substrate. In any case, full protection against chemical
modification was shown to be possible by the high affinity substrate
(S)-citramalate.
In conclusion, the combination of the kinetic characteristics of the
mutants and the chemical modification and substrate protection against
chemical modification strongly suggest a direct interaction between
Arg-425 and the carboxylate at the RS groups of the divalent substrates. The positive charge of Arg-425 seems to be
important in this interaction, but the chemistry of the guanidinium
groups may play a role as well. Although Arg-425 is not essential for
transport by CitP, its role in specifically interacting with the second
carboxylate of di- and tricarboxylate substrates makes it essential for
the generation of the membrane potential which is the physiological
function of CitP.
. CitP has a high affinity for
substrates that have a "second" carboxylate at one of the
R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J. S. (1999)
Biochemistry 38, 10352-10360). Monovalent anionic
substrates that lack this second carboxylate were found to bind
with a low affinity. In the present study we have constructed
site-directed mutants, changing Arg-425 into a lysine or a cysteine
residue. By using two substrates, i.e.
(S)-malate and 2-hydroxyisobutyrate, the substrate
specificity of the mutants was analyzed. In both mutants the affinity
for divalent (S)-malate was strongly decreased, whereas the
affinity for monovalent 2-hydroxyisobutyrate was not. The largest
effect was seen when the arginine was changed into the neutral
cysteine, which reduced the affinity for (S)-malate over
50-fold. Chemical modification of the R425C mutant with the sulfhydryl
reagent 2-aminoethyl methanethiosulfonate, which restores the positive
charge at position 425, dramatically reactivated the mutant
transporter. The R425C and R425K mutants revealed a substrate
protectable inhibition by other sulfhydryl reagents and the lysine
reagent 2,4,6-trinitrobenzene sulfonate, respectively. It is concluded
that Arg-425 complexes the charged carboxylate present in divalent
substrates but that is absent in monovalent substrates, and thus plays
an important role in the generation of the membrane potential.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), in combination with a high
promiscuity toward the two R groups (6). In fact, next to
the physiological substrates, CitP is able to transport a wide range of
2-hydroxycarboxylates containing various R groups. The
ability to accept both a neutral as well as a negatively charged
R group is the basis for membrane potential generation by
CitP. CitP is known to have a high affinity for divalent di- and
tricarboxylates like citrate and (S)-malate, which have a
"second" carboxylate at one of the R groups (6). Monocarboxylates like lactate and 2-hydroxyisobutyrate
(2-HIB),1 which lack this
second carboxylate, were found to bind with a low affinity but were
still transported efficiently. The high affinity of CitP for substrates
with a second carboxylate was explained by postulating a strong,
possibly electrostatic, interaction between the protein and the second
carboxylate. Positively charged residues such as arginine, lysine, or
histidine were thought to be able to participate in such an interaction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
Membrane topology and amino acid sequence of
the C-terminal part of CitP. The topology model is based on
studies of the Na+-dependent citrate
transporter CitS of K. pneumoniae which is homologous to
CitP (11). Conserved residues in the amino acid sequence alignment of
the 2-HCT family (8) were indicated in gray.
View larger version (34K):
[in a new window]
Fig. 2.
Expression levels of CitP, R425K, and
R425C. RSO membrane vesicles prepared from L. lactis
NZ9000-expressing CitP (lane 1), R425K (lane 2),
and R425C (lane 3) were analyzed by Western blotting using
antibodies raised against the His tag. The position of the molecular
mass markers was indicated on the right.
)). Only when measured for a
prolonged period (Fig. 3C) significant efflux could be
observed. The rate of efflux seemed not significantly different for the
wild type and mutant transporters. Dilution of the membranes containing
the wild type transporter in buffer containing 5 mM
unlabeled (S)-malate resulted in very rapid release of the
label from the membranes due to CitP-mediated exchange (Fig.
3A (
)). Exchange catalyzed by the R425K and R425C mutant
was considerably slower but significantly faster than efflux (Fig. 3,
B and C ()). The Arg to Cys mutation had a
more drastic effect on the exchange rate than the conservative Arg to
Lys mutation.
View larger version (14K):
[in a new window]
Fig. 3.
Homologous (S)-malate
exchange catalyzed by CitP, R425K, and R425C. RSO membrane
vesicles prepared from L. lactis NZ9000-expressing CitP
(A), R425K (B), and R425C (C) were
preloaded with 5 mM
(S)-[14C]malate and diluted 100-fold into
buffer containing 5 mM (S)-malate ( ) or no
additions ().
View larger version (12K):
[in a new window]
Fig. 4.
CitP substrates. Chemical structure of
the substrates of CitP used in this study. The R groups are
defined as RR and RS to
discriminate between the different positions of the R groups
in chiral compounds. Inset, the 2-hydroxycarboxylate
motif.
View larger version (15K):
[in a new window]
Fig. 5.
Affinity of CitP (A), R425K
(B), and R425C (C) for
(S)-malate and 2-HIB. RSO membrane vesicles
prepared from L. lactis NZ9000-expressing CitP
(A), R425K (B), or R425C (C) were
preloaded with 5 mM
(S)-[14C]malate and diluted 100-fold into
buffer containing the indicated concentrations of (S)-malate
( ) or 2-HIB (
). The curves were analyzed as described
under "Experimental Procedures," and the obtained
Kmapp and
Vmax values are given in Table I. Expression
level for CitP was lower than for R425K and R425C (see text).
Kinetic parameters for CitP, R425K, and R425C in exchange
,
N(CH3)3+, or
NH3+ for MTSES, MTSET, and MTSEA,
respectively. Right-side-out membrane vesicles containing the
transporters were treated with the MTS reagents, and
(S)-malate exchange was measured using non-saturating external (S)-malate concentrations (21). Wild type CitP
contains one Cys residue at position 361 in putative TMS X.
Treatment of RSO membranes containing CitP with the MTS reagents had no
effect on the exchange activity (Table
II). Apparently Cys-361 is not accessible
to the reagents or the modification does not affect activity. In
contrast, exchange catalyzed by the R425C mutant was severely affected
indicating a specific modification of the Cys at position 425 (Table
II). The most striking effect was seen upon incubation with MTSEA which
restores a positive charge at position 425 (Fig.
6). The homologous exchange rate
increased about 50-fold under the conditions of the experiment. In
fact, the exchange catalyzed by MTSEA-treated R425C was faster than observed for the R425K mutant (compare Figs. 6 and 3B). In
contrast to MTSEA, the more bulky positively charged reagent MTSET and the negatively charged MTSES reduced the (S)-malate
transport activity of the mutant to undetectable levels. The results
indicate that the introduction of a small positive group at position
425 in the R425C mutant can largely restore exchange activity.
Exchange (% of control) catalyzed by R425C and CitP after treatment
with sulfhydral reagents
View larger version (13K):
[in a new window]
Fig. 6.
Modification of R425C by MTSEA. RSO
membrane vesicles prepared from L. lactis NZ9000-expressing
R425C were incubated for 10 min in the presence ( 
and ) or
absence ( and ) of 1 mM MTSEA. After washing, the
membranes were preloaded with 5 mM
(S)-[14C]malate and diluted 100-fold into
buffer containing 5 mM (S)-malate ( and )
or no additions ( and ).
View larger version (53K):
[in a new window]
Fig. 7.
TNBS treatment of R425K
(A) and CitP (B). RSO
membrane vesicles prepared from L. lactis NZ9000-expressing
R425K and CitP were incubated for 2 h with 5 mM TNBS
at the indicated pH. After washing, the vesicles were loaded with 5 mM (S)-[14C]malate and diluted
100-fold into buffer containing unsaturating (S)-malate
concentrations, i.e. 1.5 mM
(S)-malate in the case of R425C and 0.15 mM in
the case of CitP. Exchange rates were given as a percentage of the
exchange rate in the untreated membranes. Error bars
represent the S.D.
View larger version (57K):
[in a new window]
Fig. 8.
Substrate protection against modification by
pCMB (A) and TNBS (B).
A, RSO membrane vesicles containing R425C were incubated for
0.5 min with 0.1 mM pCMB in the presence or absence of the
indicated concentrations of 2-HIB or butyrate. After washing, the
membranes were preloaded with 5 mM
(S)-[14C]malate and diluted 100-fold into
buffer containing 5 mM (S)-malate. B,
RSO membrane vesicles containing R425K were incubated for 2 h with
30 mM TNBS at pH 7 in the presence or absence of 60 mM (S)-malate, (S)-citramalate, or
succinate. After washing, the vesicles were preloaded with 5 mM (S)-[14C]malate and diluted
100-fold into buffer containing 1.5 mM
(S)-malate. Exchange rates were given as a percentage of the
exchange rate without addition of TNBS. Error bars represent
the S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, in
which the R groups can vary. These R groups were
defined as RR and RS to
discriminate between different positions of the R groups
around the asymmetric C-2 atom in chiral substrates (Fig. 4,
inset). CitP is known to have a high affinity for substrates
that have a second charged carboxylate at the RS position, like citrate and (S)-malate (6). Monovalent
substrates that lack this second carboxylate, like lactate and 2-HIB,
were found to bind with low affinity but were still transported
efficiently. A non-essential interaction between the protein and the
second carboxylate of the divalent substrates was postulated that would increase affinity for these substrates. Arg-425 was selected to be a
good candidate for the site on the protein involved in this interaction
because of the following. (i) Arg-425 is located in the stretch of 46 residues at the C terminus, which has been suggested to be involved in
the interaction with the R groups (8). (ii) Arg-425 is
located in a transmembrane segment of the transporter. (iii) Arg-425 is
conserved in the 2-HCT family (8).
| |
ACKNOWLEDGEMENT |
|---|
We thank W. N. Konings for carefully reading the manuscript and many helpful suggestions.
| |
FOOTNOTES |
|---|
* 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.
Supported by a grant from the Netherlands Organization for
Scientific Research.
§ To whom correspondence should be addressed: Dept. of Microbiology, University of Groningen, Biological Centre, Kerklaan 30, 9751 NN Haren, The Netherlands. Tel.: 31-50-3632155; Fax: 31-50-3632154; E-mail: j.s.lolkema@biol.rug.nl.
Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.M005940200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: 2-HIB, 2-hydroxyisobutyrate; RSO, right-side-out; pCMB, p-chloromercuribenzoic acid; pCMBS, p-chloromercuribenzosulfonate; TNBS, 2,4,6-trinitrobenzene sulfonate; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; TMS, transmembrane segment; 2-HCT, 2-hydroxycarboxylate transporter.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Lolkema, J. S., Poolman, B., and Konings, W. N. (1996) in Handbook of Biological Physics (Konings, W. N. , Kaback, H. R. , and Lolkema, J. S., eds) , pp. 229-260, Elsevier/North-Holland Biomedical Press, Amsterdam |
| 2. | Bandell, M., Lhotte, M. E., Marty Teysset, C., Veyrat, A., Prevost, H., Dartois, V., Divies, C., Konings, W. N., and Lolkema, J. S. (1998) Appl. Environ. Microbiol. 64, 1594-1600 |
| 3. | Marty-Teysset, C., Lolkema, J. S., Schmitt, P., Divies, C., and Konings, W. N. (1995) J. Biol. Chem. 270, 25370-25376 |
| 4. | Marty Teysset, C., Posthuma, C., Lolkema, J. S., Schmitt, P., Divies, C., and Konings, W. N. (1996) J. Bacteriol. 178, 2178-2185 |
| 5. | Magni, C., de Mendoza, D., Konings, W. N., and Lolkema, J. S. (1999) J. Bacteriol. 181, 1451-1457 |
| 6. | Bandell, M., and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360 |
| 7. | Bandell, M., Ansanay, V., Rachidi, N., Dequin, S., and Lolkema, J. S. (1997) J. Biol. Chem. 272, 18140-18146 |
| 8. | Bandell, M., and Lolkema, J. S. (2000) Biochemistry 39, 13059-13067 |
| 9. | van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589 |
| 10. | van Geest, M., Nilsson, I., von Heijne, G., and Lolkema, J. S. (1999) J. Biol. Chem. 274, 2816-2823 |
| 11. | van Geest, M., and Lolkema, J. S. (1999) J. Biol. Chem. 274, 29705-29711 |
| 12. | De Ruyter, P.-G.-G.-A., Kuipers, O.-P., and De Vos, W.-M. (1996) Appl. Environ. Microbiol. 62, 3662-3667 |
| 13. | Karlin, A., and Akabas, M. H. (1998) Methods Enzymol. 293, 123-145 |
| 14. | Kuipers, O. P., Beerthuyzen, M. M., Siezen, R. J., and De Vos, W. M. (1993) Eur. J. Biochem. 216, 281-291 |
| 15. | Putman, M., van Veen, H. W., Poolman, B., and Konings, W. N. (1999) Biochemistry 38, 1002-1008 |
| 16. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2 Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 17. | Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 61-68 |
| 18. | Holo, H., and Nes, I. F. (1989) Appl. Environ. Microbiol. 55, 3119-3123 |
| 19. | Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 |
| 20. | Akabas, M. H., Stauffer, D. A., Xu, M., and Karlin, A. (1992) Science 258, 307-310 |
| 21. | Ramamoorthy, S., Melikian, H. E., Qian, Y., and Blakely, R. D. (1998) Methods Enzymol. 296, 347-370 |
| 22. | Fields, R. (1972) Methods Enzymol. 25, 464-468 |
| 23. | Grubmeyer, C., Segura, E., and Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304 |
| 24. | Olami, Y., Rimon, A., Gerchman, Y., Rothman, A., and Padan, E. (1997) J. Biol. Chem. 272, 1761-1768 |
| 25. | Mitchell, J. B., Thornton, J. M., Singh, J., and Price, S. L. (1992) J. Mol. Biol. 226, 251-262 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  W. A. M. Wolken, P. M. Lucas, A. Lonvaud-Funel, and J. S. Lolkema The Mechanism of the Tyrosine Transporter TyrP Supports a Proton Motive Tyrosine Decarboxylation Pathway in Lactobacillus brevis J. Bacteriol., March 15, 2006; 188(6): 2198 - 2206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Sobczak and J. S. Lolkema The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism Microbiol. Mol. Biol. Rev., December 1, 2005; 69(4): 665 - 695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Sobczak and J. S. Lolkema Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae J. Biol. Chem., July 23, 2004; 279(30): 31113 - 31120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. N. Kastner, M. Prummer, B. Sick, A. Renn, U. P. Wild, and P. Dimroth The Citrate Carrier CitS Probed by Single-Molecule Fluorescence Spectroscopy Biophys. J., March 1, 2003; 84(3): 1651 - 1659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. P. Krom, R. Aardema, and J. S. Lolkema Bacillus subtilis YxkJ Is a Secondary Transporter of the 2-Hydroxycarboxylate Transporter Family That Transports L-Malate and Citrate J. Bacteriol., October 15, 2001; 183(20): 5862 - 5869. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
