(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
[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.
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. 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.
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 ()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).
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 ()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.
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 [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.
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 (,
,
) were placed in 100 mM K
SO
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
(
).
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 (,
,
) 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.
The presence of an aspartate-linked
antiporter was also supported by an analysis of how steady state levels
of [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.
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 [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) ).
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) of 0.36
± 0.03 mM and a maximal velocity of 0.40 ± 0.03
µmol/min/mg of protein (means ± S.E.).
Figure 3:
Kinetic analysis of aspartate
self-exchange. In the experiment of Fig. 2B, the
kinetic parameters of [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.
We also obtained a
direct measurement of the dissociation constant, K(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,
Figure 4:
Substrate protection of solubilized AspT.
A detergent extract (342 µg of protein/ml) was placed at 37 °C
in the absence () 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.
where (S) represents the aspartate concentration used
for stabilization. For the aspartate levels tested here (2.5-20
mM), this relationship suggests a K(aspartate) of 0.3 ± 0.02 mM (Fig. 4, legend).
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 [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
.
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.
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.
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 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.
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 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.
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, 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) .