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(Received for publication, June 12, 1995; and in revised form, November 8, 1995) From the
We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive
metabolic cycle by using reconstitution to monitor the activity of the
carrier, termed AspT, expected to carry out the electrogenic exchange
of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl
glucoside in the presence of 0.4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to
prepare proteoliposomes loaded with either aspartate or alanine.
Aspartate-loaded proteoliposomes accumulated external
[
Nutrient transport by bacteria is usually thought of as
consuming metabolic energy, since this step is typically driven by an
ion-motive gradient (e.g. Such precedents suggest a new way of
interpreting the relationship between anion transport and
decarboxylation reactions in microorganisms. For example, some strains
of the Lactobacilli catalyze the decarboxylation of either L-aspartate ( As their central element, proton-motive metabolic cycles
have a vectorial component(s) that mediates the electrogenic exchange
of precursor and product. Accordingly, the specific goal of work
reported here was to determine whether this transport reaction is found
in Lactobacilli subsp. M3, a cell which readily converts
aspartate to alanine by intracellular decarboxylation. To approach this
issue, we used reconstitution of membrane protein as an analytical tool
to probe for the expected exchange of aspartate and alanine in
proteoliposomes. Our experiments document that membranes of Lactobacilli subsp. M3 display an electrogenic
aspartate:alanine exchange of the sort required by a proton-motive
metabolic cycle. This precursor:product antiport is catalyzed by a
single element, termed ``AspT'' (for aspartate transporter),
which catalyzes both the homologous self-exchange of aspartate and the
heterologous antiport of aspartate and alanine.
Reconstitution was in a final volume
of 1 ml, using 400 µl of detergent extract (or control lipid
extract), 130 µl of bath-sonicated liposomes (5.9 mg of E. coli phospholipid), 18 µl of 15% octylglucoside, with the balance
comprised of 100 mM phosphate (pH 7) as the potassium or NMG (
A simplified assay (13, 14) was
used to monitor AspT activity during tests of its stabilization, in
vitro, by substrate. In these experiments, a detergent extract
(250-450 µg of protein/ml) was placed at 37 °C, along
with desired additives. To quench the reaction, a 100-µl aliquot
was removed and placed in a chilled tube containing 100 µl of 20
mM potassium aspartate and other components required for
reconstitution (above), using amounts scaled to a final volume of 250
µl. Bath-sonicated liposomes (1.36 mg) were added after 5 min, and
the mixture was allowed to remain on ice for 20 min before adding 5 ml
of a solution of 100 mM potassium phosphate (pH 7) plus 100
mM potassium aspartate to form aspartate-loaded
proteoliposomes. To assess transport, duplicate 0.2-ml aliquots were
then applied, under vacuum, to the center of GSTF Millipore filters
(0.22-µm pore size). External aspartate was removed by two 5-ml
rinses with assay buffer, and after release of the vacuum, the reaction
was initiated by overlaying proteoliposomes, on the filter, with 0.3 ml
of this same buffer containing 100 µM [
Figure 1:
Reconstitution of aspartate
self-exchange. Proteoliposomes and liposomes were loaded with 100
mM potassium aspartate plus 100 mM potassium
phosphate (pH 7) and washed and resuspended as described under
``Experimental Procedures.'' To start the experiment,
proteoliposomes (
Figure 5:
The electrogenic nature of
aspartate:alanine exchange. Proteoliposomes or liposomes were loaded
with 100 mM alanine plus 100 mM phosphate (pH 7) as
either the NMG (
The presence of an aspartate-linked
antiporter was also supported by an analysis of how steady state levels
of [
Figure 2:
Distribution of aspartate between internal
and external pools. A, proteoliposomes loaded with 100 mM potassium phosphate (pH 7) and potassium aspartate at the
indicated concentrations were suspended at 3-5 µg of
protein/ml and exposed to 100 µM [
We performed two additional experiments to
characterize more directly the interaction between AspT and its
substrate, aspartate. In one case, we undertook a simple kinetic study,
relying on samples filtered after 10 s to estimate initial velocities (Fig. 3). In that experiment, we found that the self-exchange
reaction had a Michaelis constant (K
Figure 3:
Kinetic analysis of aspartate
self-exchange. In the experiment of Fig. 2B, the
kinetic parameters of [
We also obtained a
direct measurement of the dissociation constant, K
Figure 4:
Substrate protection of solubilized AspT.
A detergent extract (342 µg of protein/ml) was placed at 37 °C
in the absence (
where (S) represents the aspartate concentration used
for stabilization. For the aspartate levels tested here (2.5-20
mM), this relationship suggests a K
In Lactobacillus subsp. M3, the inhibitor
sensitivity of ATP synthesis associated with aspartate metabolism
suggests operation of a proton-motive metabolic cycle (Table 1)
involving the exchange of the precursor, aspartate, and its
decarboxylation product, alanine. To test this idea we used
reconstitution to characterize [ The diagram of Fig. 6summarizes the proposed proton-motive cycle in Lactobacillus subsp. M3 and in other cells that use
decarboxylation to convert aspartate into alanine and CO
Figure 6:
A proton-motive metabolic cycle associated
with aspartate decarboxylation. Two possible scenarios are shown. Part A (left) outlines the steady state operation
of a proton-motive metabolic cycle based on the function of AspT. For
this case, inward transport of aspartate
Our experiments indicate this
thermodynamic proton pump (Fig. 6) will characterize the steady state during aspartate metabolism by Lactobacillus subsp. M3. On the other hand, the discrepancy between the affinity
of AspT for aspartate and alanine (0.3 mM and Analysis of anion
transport and exchange in O. formigenes provided the first
example of a proton-motive metabolic cycle(3, 4) , and
subsequent work (8, 9, 10, 11, 23) has
identified additional cycles in both Gram-negative and Gram-positive
forms (reviewed in Refs. 7 and 24). For the most part, these examples
are linked to decarboxylations (e.g.Fig. 6)(3, 8, 9, 10, 11, 23) ,
although it has been clear that more complex metabolic ensembles may be
similarly structured. Indeed, among the lactic acid bacteria one now
finds useful models of both types. Thus, there is evidence that the
processing of malate (8, 9, 10) ,
histidine(11) , and aspartate (this work) offer cases in which
a simple metabolic sequence is arranged so as to generate a
proton-motive force by combining physically separated vectorial and
scalar events. A more complex, ensemble model is found in Leuconostoc oenos(23) , where entry of the anion,
citrate
Volume 271,
Number 6,
Issue of February 9, 1996 pp. 3079-3084
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
MECHANISM FOR DEVELOPMENT OF A PROTON-MOTIVE FORCE IN BACTERIA (*)
H]aspartate by exchange with internal substrate;
this homologous self-exchange (K
= 0.4
mM) was insensitive to potassium or proton ionophores and was
unaffected by the presence or absence of Na,
K
, or Mg
. Alanine-loaded
proteoliposomes also took up [
H]aspartate in a
heterologous antiport reaction that was stimulated or inhibited by an
inside-positive or inside-negative membrane potential, respectively.
Several lines of evidence suggest that these homologous and
heterologous exchange reactions were catalyzed by the same functional
unit. Thus, [H]aspartate taken up by AspT during
self-exchange was released by a delayed addition of alanine. In
addition, the spontaneous loss of AspT activity that occurs when a
detergent extract is held at 37 °C prior to reconstitution was
prevented by the presence of either aspartate (K(aspartate) = 0.3 mM)
or alanine (K
(alanine)
10
mM), indicating that both substrates interact directly with
AspT. These findings are consistent with operation of a proton-motive
metabolic cycle during aspartate metabolism by Lactobacillus
subsp. M3.
t;ex2html_html_special_mark_amp;mgr; or
t;ex2html_html_special_mark_amp;mgr;) or by hydrolysis
of a phosphoester bond (e.g. ATP or
PEP)(1, 2) . Recently, however, a new class of
nutrient transport reactions has been identified, one in which
substrate transport is actually used to generate rather than consume
energy. The first and best understood of these reactions is found in Oxalobacter formigenes(3, 4) , a
Gram-negative, obligate anaerobe that exploits the decarboxylation of
oxalate to support transmembrane ion-motive gradients(5) . This
cell mediates the exchange of divalent oxalate with the product of its
intracellular decarboxylation, monovalent formate(6) , using a
membrane transporter named OxlT(4) . The one-for-one exchange
of oxalate
and formate
polarizes
the membrane (electrically negative, inside), while the decarboxylation
reaction serves to generate an internal alkalinity, since a single
cytosolic proton is consumed during production of formate. As a result,
the metabolic sequence, oxalate entry, oxalate decarboxylation, formate
exit, acts as a proton pump (3) or ``proton-motive
metabolic cycle'' ( (3) and (4) ; reviewed in (7) ). In the same way and in other bacteria, the transport
(vectorial) and decarboxylation (scalar) reactions associated with
conversion of malate to lactate (8, 9, 10) or
histidine to histamine (11) have been shown to act as
proton-motive metabolic cycles.
)or L-glutamate, (
)with a near-stoichiometric release of the products, L-alanine or -aminobutyrate (and CO
),
respectively. These decarboxylations support ATP
synthesis in a manner consistent with the idea that processing of these
anions involves a proton-motive metabolic cycle
(and see
below).
Organism, Growth Conditions, and Preparation of
Membrane Vesicles
Lactobacillus subsp. M3 was grown
under anaerobic conditions at 30 °C in MRS broth (Difco)
supplemented with 30 mML-aspartate and 5% sodium
chloride. After growth to stationary phase (3 days), cells were
harvested by centrifugation, washed with 100 mM potassium
phosphate (pH 7), and membrane vesicles were prepared by high pressure
lysis in the presence of 100 mM potassium phosphate (pH 7), as
described (12) ; vesicles were stored at -70 °C as a
concentrated stock (10-20 mg/ml protein).
Solubilization and Reconstitution of AspT
Membrane
vesicles (1-2 mg of protein) were solubilized(12) , using
1.25% (w/v) octylglucoside in the presence of 0.4% (w/v) acetone/ether
washed Escherichia coli phospholipid, 100 mM potassium phosphate (pH 7), 4 mM MgSO
, 1
mM dithiothreitol, 0.75 mM phenylmethylsulfonyl
fluoride, and 20% glycerol. Control extracts were prepared in the same
way, but without added protein.)salt, and 1 mM dithiothreitol. After 20 min on
ice, proteoliposomes (or control liposomes) were formed at 23 °C by
rapid injection into 20 ml of a loading buffer containing 100 mM potassium phosphate (pH 7) and 1 mM dithiothreitol, along
with 25-150 mM aspartate (potassium, or NMG salts, as
specified). After a further 20 min, the substrate-loaded
proteoliposomes (or liposomes) were recovered by centrifugations and
washings(12) , with resuspension in 100 mM potassium
or NMG sulfate plus 100 mM potassium or NMG-phosphate (pH 7)
and 1 mM dithiothreitol. The final resuspension volume was
usually 300 µl, giving protein and lipid at 50-250
µg/ml and 13 mg/ml, respectively(12) . When proteoliposomes
(liposomes) were loaded with 100 mM alanine, the same
procedure was followed except that the buffer for washing and
resuspension was the same as the loading buffer, and the resuspension
volume was reduced to 80 µl.
Assays of Transport
For assay of
[H]aspartate transport by aspartate-loaded
particles, proteoliposomes were diluted 20-fold from their concentrated
stock into an appropriate volume of assay buffer (resuspension buffer
lacking dithiothreitol) along with other required materials. After a
1-3-min preincubation at 23 °C, labeled substrate was added
to a nominal concentration of 100 µM, and at the required
times aliquots of 100 µl, corresponding to about 0.065 µl
internal volume (12) , were removed for membrane filtration
(0.22-µm pore sized GSTF Millipore filters), followed by two washes
with 5 ml of assay buffer (12) . For transport assays of
alanine-loaded proteoliposomes, or those prepared at pH 8,
proteoliposomes were diluted 133-fold into assay buffer containing
labeled substrate.H]aspartate. The reaction was terminated by
vacuum filtration after the exchange reaction had reached its steady
state (10 min); this was followed by three quick rinses with buffer to
remove residual external radioactivity.Other Assays
Protein was measured using a
modification of method of Schaffner and Weissmann(15) . ATP in
boiled cell extracts was determined with the firefly assay, as
described by Mason et al.(16) .Chemicals
L-[2,3-H]Aspartate
acid (26.3 Ci/mmol) was purchased from DuPont NEN Corp. Phospholipid
was purified from the crude E. coli lipid provided by Avanti
Polar Lipids, Inc.(12) . Octylglucoside was from Boehringer
Mannheim.
Aspartate-dependent ATP Production by Intact
Cells
In Gram-positive anaerobes such as the Lactobacilli, the
proton-coupled FF
-ATPase hydrolyzes ATP made
during fermentation of sugars or amino acids to generate a
proton-motive force. As a consequence, ATP levels do not change when
such cells are treated with an ATPase inhibitor (e.g. DCCD) or
with proton ionophores (e.g. CCCP, or the combination of
nigericin and
valinomycin)(17, 18, 19, 20) . It
was unexpected, therefore, that these inhibitors strongly reduced ATP
synthesis associated with the decarboxylation of aspartate and
production of alanine plus carbon dioxide in Lactobacillus subsp. M3 (Table 1). By contrast, this inhibitor sensitivity
is understandable if aspartate decarboxylation is organized as a
proton-motive metabolic cycle (3, 4, 7) involving the antiport of precursor
(aspartate) and product (alanine), since in that case ATP synthesis
would arise from reversal of the ATPase. With this possibility in mind,
we used reconstitution of membrane protein to probe for the presence of
a carrier which could mediate the required antiport reaction.
Identification and Characterization of the Aspartate
Self-exchange Reaction
To identify the putative
aspartate:alanine exchange carrier, AspT, our initial focus was a study
of aspartate-loaded proteoliposomes, with the expectation that AspT
would display an aspartate self-exchange. For this reason,
proteoliposomes were loaded with 100 mM aspartate and
suspended in a sulfate-based medium to which 100 µM external [H]aspartate was added. For these
general conditions, we found substantial transport of the labeled
substrate. In the experiment shown (Fig. 1), the steady state
incorporation of [H]aspartate was approximately
500 nmol/mg of protein. Assuming [H]aspartate had
been taken up uniformly by all proteoliposomes (but see below), this
corresponded to a 30-fold accumulation of substrate over its
concentration in the medium. Moreover, the incorporated material was
readily chased by a later addition of excess unlabeled aspartate, as
expected if [H]aspartate had been taken up by an
exchange reaction. And since accumulated
[H]aspartate was released by either aspartate or
alanine (Fig. 1), it seemed feasible that both compounds served
as substrates. No accumulation of [H]aspartate
was observed when liposomes (no protein) were tested (Fig. 1),
nor did aspartate accumulate in proteoliposomes in which internal
sulfate replaced aspartate (not shown in Fig. 1; see Fig. 5). In other experiments of this sort we found transport of
[H]aspartate to be unaffected by the presence of
the potassium ionophore, valinomycin (1 µM), or by
addition of the protonophore, carbonyl cyanide
carboxymethoxyphenylhydrazone (1 µM) (data not shown). Nor
was [H]aspartate transport influenced by
substitution of sodium for potassium or by the presence of
Mg or EDTA (each at 10 mM; data not given).
Based on these observations, we concluded that membranes of Lactobacillus subsp. M3 contain a carrier capable of mediating
an aspartate self-exchange, as expected of AspT, and that this reaction
is an electroneutral event operating independently of coupling cations
such as Na
, K
, Ca
,
or Mg
.
, 
, 
) were placed in 100 mM KSO plus 100 mM potassium P
(pH 7) at 3.3 µg of protein/ml and given 100 µM [H]aspartate; an equivalent volume of
liposomes () was treated in the same way. To estimate substrate
transport, aliquots were taken at the times indicated for filtration
and washing. The arrow denotes addition of 15 mM unlabeled aspartate (
) or 30 mM unlabeled alanine
().
, 
, ) or potassium (
) salts.
To start the experiment, proteoliposomes (or liposomes) suspended at
1.9 mg of protein/ml in their respective loading buffers were diluted
133-fold into assay media containing 100 µM [H]aspartate along with 100 mM SO plus 100 mM phosphate as the potassium
() or NMG (, , ) salts; with one exception
(
), 1 µM valinomycin (Val) was also
present. Control liposomes and proteoliposomes were assayed using the
potassium-based medium, with valinomycin. These controls included
liposomes prepared with either NMG or potassium salts of alanine and
proteoliposomes containing 75 mM NMG sulfate rather than 100
mM NMG alanine; because these controls gave no significant
aspartate transport, they are not individually noted in the figure for
reasons of clarity. Aspartate transport was determined for all
combinations of potassium or NMG alanine-loaded proteoliposomes using
potassium- or NMG-based assay media, with and without valinomycin; data
not shown here are included in Table 2.
H]aspartate accumulation were influenced by
the relative sizes of the internal and external aspartate pools. For
example, [H]aspartate incorporation increased in
direct proportion to elevation of internal substrate concentration (Fig. 2A). We also addressed this issue quantitatively
by experiments in which [H]aspartate transport
was monitored as the external pool was expanded by known amounts (Fig. 2B). This led to predictably increased ratios of
external to internal [H]aspartate and that
relationship was used to calculate an aspartate-accessible internal
mass of 5.3 ± 1.3 nmol/mg lipid (Fig. 2, legend). Given
an overall internal volume of about 1 µl/mg lipid (12, 21) and the internal aspartate concentration of
100 mM, the total internal aspartate pool should be about 100
nmol/mg lipid(12) . Therefore, the observed accessible mass
(5.3 nmol/mg lipid) indicates that only a small fraction (5%) of
proteoliposomes carried out the aspartate self-exchange reaction. This
level of activity was typical of the work reported here, and for this
reason, presuming a random distribution of AspT among proteoliposomes,
we concluded that no single proteoliposome contained more that one
functional unit of AspT.
H]aspartate. Steady state incorporation of
labeled aspartate was measured after 10 min. B, in a separate
experiment, proteoliposomes (11 µg of protein/ml) were loaded with
100 mM aspartate, as in Fig. 1, and given 100
µM [H]aspartate and unlabeled
aspartate to arrive at the final concentration indicated on the
abscissa. In five replicate trials, samples were taken after 10 min to
determine the ratio of [H]aspartate in the medium
to that in proteoliposomes, as given on the ordinate. Inset,
an enlargement of the scale. Assuming isotope equilibrium, the
horizontal intercept gives the residual external aspartate (22 nmol/ml)
brought into the assay along with proteoliposomes, while the reciprocal
of the slope of the line gives the internal
[H]aspartate-accessible pool (3.6 nmol/ml) (see (22) ). Assuming an internal volume of 1 µl/mg lipid (12, 21) and the known internal aspartate
concentration of 100 mM, this accessible pool corresponds to
an internal volume of 0.053 µl/mg phospholipid (see text and (3) and (22) ).
) of 0.36
± 0.03 mM and a maximal velocity of 0.40 ± 0.03
µmol/min/mg of protein (means ± S.E.).
H]aspartate transport were
determined by sampling after 10 s to estimate initial velocities.
Substrate concentrations were corrected for the residual external
aspartate carried into the assay from the proteoliposome stock (Fig. 2B, legend). Inset, a linear transform
was used to calculate the Michaelis constant (K)
and maximal velocity (V
). The data are means of
triplicate measurements.
(aspartate), by monitoring the kinetics with
which aspartate stabilized solubilized AspT. When a detergent extract
was placed at 37 °C, reconstitution at periodic intervals showed
that recoverable AspT activity decayed in an exponential fashion, with
a half-life of about 10 s (0.15 min) (Fig. 4). Added substrate
was clearly protective, and AspT lifetime was increased roughly 75-fold
by the presence of 20 mM substrate (half-life of 11 min; Fig. 4, legend). This stabilization is presumed to reflect the
binding of substrate by AspT, with generation of liganded complex more
resistant to denaturation, as noted for OxlT (14) . Provided
that liganded AspT is very much more stable than the unliganded
carrier, a realistic view given the observed response to excess
aspartate (Fig. 4), the stabilization achieved by substrate can
be analyzed quantitatively to derive the dissociation constant of the
AspT-aspartate complex(14) . In such cases, the -fold increase
in AspT lifetime (R) is related to K(aspartate) by the following
expression,
) and presence of aspartate at 2.5 mM (), 5 mM (), 10 mM (), and
20 mM (). To follow the decay of AspT, aliquots were
removed at the indicated times and placed in chilled quench tubes
containing 20 mM aspartate along with the other components
required for later reconstitution. After reconstitution,
proteoliposomes were tested for residual AspT activity by the
abbreviated assay (see ``Experimental Procedures''). In the
absence of added substrate, recoverable AspT activity disappeared with
a half-life of 0.15 min. Aspartate at 2.5 mM, 5 mM,
10 mM, and 20 mM gave half-lives of 1.25, 2.7, 5.5,
and 11 min, respectively, and from (see text), these
predicted corresponding K
(aspartate)
values of 0.34, 0.29, 0.28 and 0.28
mM.
(aspartate) of 0.3 ± 0.02 mM (Fig. 4, legend).Heterologous Exchange Catalyzed by AspT
Intact
cells of Lactobacillus subsp. M3 convert aspartate to alanine plus CO, and if this reflects operation of a
proton-motive metabolic cycle, it seemed possible that AspT would carry
out the heterologous exchange of aspartate and alanine. This was
implied by the finding that substrate taken up during aspartate
self-exchange was released by added aspartate or alanine (Fig. 1). Since conversion of aspartate to alanine is very
nearly stoichiometric in Lactobacillus subsp. M3, one further expects the aspartate:alanine exchange ratio to be a
one-for-one antiport, and for that case one anticipates that the
reaction will be electrogenic, since near physiological pH (pH 7) the
net charge on aspartate is -1 (pK(COOH)
= 2.09, pK(NH)
= 9.82, pK
(COOH) = 3.86), while that
on alanine is 0 (pK(COOH) = 2.34,
pK(NH) =
9.69). This view was examined in studies of aspartate transport by
alanine-loaded proteoliposomes, using valinomycin along with external
or internal potassium to generate a membrane potential that should
accelerate or retard such an electrogenic reaction. In such experiments (Fig. 5), control proteoliposomes were prepared and assayed
using NMG as the internal and external cation. These controls, whose
behavior was largely unaffected by valinomycin, took up
[
H]aspartate at an initial rate of about
2.2-2.8 nmol/15 s/mg of protein (Fig. 5; Table 2).
Note, however, that when proteoliposomes were made with internal
potassium, so that an internally negative potential was established in
the presence of valinomycin, there was profound inhibition of
[H]aspartate transport (from 2.7 to 0.1 nmol/15
s/mg of protein), while in the reciprocal trial, when an internally
positive potential was present, there was a stimulation of
[H]aspartate movement (from 7.9 to 15.1 nmol/15
s). In addition, the presence of external, but not internal, potassium
appeared to stimulate the initial phases of aspartate transport (from
2.7 to 7.9 nmol/15 s; Table 2), although the reasons for this are
unclear. (A similar finding was made during reconstitution of the
oxalate:formate heterologous exchange from O. formigenes(3) , but potassium dependence was not evident for the
purified OxlT protein(4) .) It seems unlikely that aspartate
moved by a separate (electrogenic) pathway, independent of alanine,
since [H]aspartate did not accumulate within
proteoliposomes loaded with sulfate instead of alanine (Fig. 5),
despite the favorable electrical gradient (positive inside). Neither
did the positive effect of membrane potential appear secondary to
development of a pH gradient (alkaline inside), since lowering internal
buffering power 4-fold had no effect on the 2-fold enhancement found
for [H]aspartate transport (not shown). We do
note that while an unfavorable membrane potential gave strong
inhibition of [H]aspartate transport, the
stimulation by a favorable electric gradient was modest (Table 2). Such an asymmetry might occur if the AspT carrier
operates near saturation under our usual conditions. Indeed, results
consistent with this idea were provided in an additional experiment
that compared results obtained when internal alanine was reduced from
100 to 30 mM. In that case, basal and stimulated rates for
proteoliposomes loaded with 100 mM alanine were comparable
with those found earlier (12 ± 0.7 and 23 ± 1.3 nmol/15
s), as was the observed 1.9-fold rate stimulation by membrane
potential. When internal alanine was reduced to 30 mM, we
recorded a lower basal rate of [H]aspartate
transport, but the same stimulated value (5.6 ± 0.7 versus 21 ± 2.1 nmol/15 s), and a correspondingly increased
(3.7-fold) stimulation by membrane potential. These supplemental
experiments, together with those described earlier ( Fig. 5and Table 2), strongly suggest that aspartate:alanine antiport is an
electrogenic reaction in which net negative charge moves in parallel
with aspartate.Alternate Substrates of AspT
Having documented
that AspT accepts both aspartate and alanine, our concluding work
centered on identification of other possible substrates, using two
different approaches. In one kind of experiment, we evaluated putative
substrates by asking whether [H]aspartate was
taken up by substrate-loaded proteoliposomes (e.g.Fig. 5). Thus, proteoliposomes were loaded with the NMG
salts of alanine, 
-alanine, glycine, or glutamate (100 mM in each case) and then suspended in the usual assay buffer (i.e. KSO plus potassium phosphate) in
the presence of 1 µM valinomycin and 100 µM [H]aspartate. In each case, proteoliposomes
accumulated labeled [H]aspartate, and in each
case a later addition of unlabeled aspartate evoked a rapid discharge
of the internalized material (not shown). A comparison of initial
velocities of [H]aspartate transport indicated an
order of preference as alanine > glycine > -alanine
glutamate (rates in the ratios of 1.0, 0.67, 0.37, and 0.04,
respectively). By contrast, [
H]aspartate
accumulation was not found for proteoliposomes prepared with internal
acetate, butyrate, formate, -aminobutyric acid, malonate, oxalate,
propionate, or pyruvate (each at 100 mM); nor did the addition
of these compounds at 30 mM affect the aspartate self-exchange
reaction (not shown). From this work, we concluded that along with
aspartate and alanine,
-alanine, glycine, and glutamate might
serve as substrates for AspT, and that AspT accepts substrates with an
amino function in the 
or , but not position. In
further work we confirmed that these alternate substrates interacted
directly with AspT by showing that their presence stabilized the
solubilized protein (experiments similar to that of Fig. 4).
Thus, when added at 20 mM, alanine, glycine, and
-alanine
gave an increase in AspT lifetime comparable with the effect of about
0.65 mM aspartate. This implies that these substrates have a
relatively low affinity for AspT (cf. ) and places
their K values at 10 mM). In similar
trials, glutamate gave some slight protection, which was not
quantitated, while -aminobutyric acid did not.
H]aspartate
transport in this cell and searched for the two reactions likely to
characterize the hypothetical antiporter, AspT: the exchange of
aspartate with itself in aspartate-loaded proteoliposomes and the
heterologous exchange of aspartate and alanine, as tested in
alanine-loaded proteoliposomes. Both reactions were demonstrable (e.g.Fig. 1and Fig. 5) for conditions in which
only a small fraction (5%) of the proteoliposomal population
contained an exchange carrier. And since
[
H]aspartate taken up by aspartate- or
alanine-loaded particles was released by adding an excess of either substrate (e.g.Fig. 1), we conclude a single
exchange carrier, AspT, carries out both homologous and heterologous
antiport reactions. Equally important, by examining the effect of
imposed electrical potential on the heterologous exchange (Fig. 5), it could be shown that negative charge moves in the
same direction as aspartate. Given the relevant pK values of the
carboxyl and amino groups on these substrates (see above), the
heterologous exchange catalyzed by AspT likely involves movement of
aspartate against alanine
; by extension,
the aspartate self-exchange is probably based on movements of
aspartate.
.
In this model, entry of negatively charged aspartate is followed by its
intracellular decarboxylation in a reaction that consumes a single
cytosolic proton. The products of this decarboxylation, CO and alanine, are assumed to leave the cell by different routes.
Owing to its small size and high lipid solubility, we presume that
CO moves outward by passive diffusion through the lipid
bilayer. Alanine, however, with its more limited capacity for passive
diffusion, requires a specific efflux pathway, and this is provided by
AspT itself. In the steady state, then, the result would be a
proton-motive cycle in which the vectorial component (AspT) catalyzes
import of a single negative charge, while the scalar reaction
(decarboxylation) ensures the stoichiometric disappearance of a single
internal proton. This association of vectorial and scalar elements
resembles that described earlier for O.
formigenes(3, 4) , but the biochemical nature of
the individual proteins differs considerably in the two organisms.
Thus, the antiporters, OxlT and AspT, are distinguished by both kinetic
behavior and substrate specificity(3, 4) , while the
decarboxylation reactions differ in their use of cofactor, coenzyme A
in O. formigenes(6) , but pyridoxal 5`-phosphate in L. subsp. M3.
is followed
by its decarboxylation and the subsequent outward movement of uncharged
alanine
. Because entry of a single negative charge (on
aspartate) is stoichiometric with consumption of a single internal
proton (during decarboxylation), these reactions comprise a
thermodynamic proton pump. Part B (right) illustrates
the alternative, pre-steady state, function suggest for AspT.
Here, an electrically neutral exchange of aspartate with OH
(or symport with H
)
allows the initial internalization of substrate, after which continued
decarboxylation can expand the internal alanine pool to a suitable
size.
10
mM, respectively) suggests a proton-motive cycle may not come
into play until internal alanine rises to an appropriately high value.
Therefore, in the pre-steady state, we suggest that AspT
mediates the electroneutral exchange of aspartate with hydroxyl ion, or
the equivalent, H/aspartate symport, (
)thereby ensuring continued influx of aspartate until
decarboxylation expands the alanine pool to a suitable size. In this
way, AspT could also provide aspartate as a substrate for conventional
metabolic pathways or for biosynthetic purposes., is eventually coupled to a steady state
proton-motive cycle by a subsequent proton-consuming metabolism. These
few precedents suggest we are at the initial stages of understanding
such emergent cycles and that it may be useful to consider wider
application of this principle in cell
biology(3, 4, 7) .
))))/aspartate
symport, the
efflux of alanine
would reverse this reaction, so that
H itself is driven outward.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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