(Received for publication, March 17, 1995; and in revised form, November 14, 1995)
From the
Uncoupling protein mediates electrophoretic transport of protons
and anions across the inner membrane of brown adipose tissue
mitochondria. The mechanism and site of proton transport, the mechanism
by which fatty acids activate proton transport, and the relationship
between fatty acids and anion transport are unknown. We used
fluorescent probes to measure H and anion transport in
vesicles reconstituted with purified uncoupling protein and carried out
a comparative study of the effects of laurate and its close analogue,
undecanesulfonate. Undecanesulfonate was transported by uncoupling
protein with a K
value similar to that
observed for laurate as it activated H
transport. Both
laurate and undecanesulfonate inhibited Cl
with
competitive kinetics. Undecanesulfonate inhibited laurate-induced
H
transport with competitive kinetics.
Undecanesulfonate and laurate differed in two important respects. (i)
Laurate caused uncoupling protein-mediated H
transport, whereas undecanesulfonate did not. (ii) Lauric acid
was rapidly transported across the bilayer by nonionic diffusion,
whereas undecanesulfonic was not. We infer that the role of uncoupling
protein in H
transport is to transport fatty acid
anions and that fatty acids induce H
transport because they can diffuse electroneutrally across the membrane.
According to this hypothesis, uncoupling protein is a pure anion porter
and does not transport protons; rather it is designed to enable fatty
acids to behave as cycling protonophores.
The mitochondrial uncoupling protein (UCP) ()is a
remarkable chemiosmotic device engineered to dissipate the protonmotive
energy of brown adipose tissue mitochondria and provide heat to the
animal. It is known that UCP-mediated uncoupling is activated by fatty
acids, and the consensus has been that UCP is a H
(or
OH
) transporter(1, 2, 3) .
It is also known that UCP, like other members of its gene family, is an
anion
transporter(1, 2, 4, 5, 6) .
Both H
and anion transport are inhibited by purine
nucleotides.
The mechanism by which FAs activate proton transport
via UCP is unknown. We have long considered UCP-mediated anion
transport to hold the key to this mystery, not least because this
property has been difficult to integrate with the physiological
function of UCP. We have recently shown that FAs inhibit Cl transport through UCP with competitive kinetics(7) ,
supporting our hypothesis that anions are transported through the FA
binding domain of UCP(8) . The close relationship between FA
interaction and anion transport permits at least two possibilities for
the mechanism of H
transport. Either FAs activate
proton transport while being anchored within the UCP (9) or FA
anions are directly transported by UCP(10, 11) . FA
anion transport by UCP would not be unexpected, in view of the fact
that UCP transports monovalent anions generally and transport rates
increase with anion hydrophobicity(5, 6) .
To
distinguish between these possibilities, we compared the properties of
laurate and its close analogue, undecanesulfonate. We measured fluxes
of H, Cl
, and K
in
proteoliposomes reconstituted with purified UCP(5) .
K
flux, in the presence of valinomycin, was used as a
measure of anionic or protonic charge flux. Both undecanesulfonate and
laurate are competitive inhibitors of UCP-mediated Cl
transport, and undecanesulfonate is a competitive inhibitor of
laurate-induced H
transport. Both anions catalyzed
charge movement across the proteoliposomal membrane, and they did so
with hyperbolic kinetics and with similar K
and V
values. In the case of laurate,
the charge movement was due to H
flux.
Undecanesulfonate did not catalyze H
flux, from which
it follows that it is transported as the anion by UCP, as are other
alkylsulfonates(6) . This difference in behavior of the two
analogues in proteoliposomes could be fully explained by their behavior
in protein-free liposomes; laurate supported rapid electroneutral
H
transport across the bilayer due to nonionic
flip-flop of the FA. Undecanesulfonate, a strong acid, was unable to
catalyze electroneutral H
transport. We conclude that
FAs cause electrophoretic H
transport in the presence
of UCP because UCP is an anion channel designed for electrophoretic
transport of FA anions. According to this mechanism, UCP-mediated
uncoupling is due to futile cycling of FAs across the inner membrane of
brown adipose tissue mitochondria. Seen in this light, UCP-transported
monovalent anions are accidental substrates of the FA anion pathway in
UCP. (
)
The data presented in
this paper are based on flux measurements of three ions. Measurement of
UCP-mediated H flux was obtained from changes in SPQ
fluorescence due to quenching by the anion of TES
buffer(12, 13) . Internal medium contained 84.4 mM TEA
SO
, 0.6 mM TEA-EGTA, and 28.8
mM TEA-TES, pH 7.2. External medium contained 60 mM K
SO
, 24.4 mM TEA
SO
, 0.6 mM TEA-EGTA, and 28.8
mM TEA-TES, pH 7.2. Measurement of UCP-dependent K
flux, reflecting the movement of ionic charge across the
membrane(14) , was obtained from changes in PBFI
fluorescence(12, 15) . Except for different probes,
internal, and external media were identical to those used for
H
flux. Measurement of UCP-mediated Cl
flux was determined from changes in SPQ fluorescence due to
Cl
quenching(5, 12) . Internal
medium contained 79.5 mM TEA
SO
, 0.6
mM TEA-EGTA, and 20 mM TRIS-PO
, pH 7.2.
External medium contained 119.25 mM KCl, 0.6 mM TEA-EGTA, and 20 mM TRIS-PO
, pH 7.2.
Figure 1:
GDP-sensitive H and
K
fluxes in the presence of undecanesulfonate and
laurate in liposomes reconstituted with UCP. Panel A,
H
efflux in the presence of laurate. The traces
obtained from SPQ fluorescence were converted to increases in
intraliposomal [TES
] (
[TES
]
) and
plotted versus time. H
flux was induced by
addition of 0.1 µM valinomycin (indicated by arrow) in the presence of 10 µM laurate. The
experiment was carried out on two separate preparations, one containing
no internal GDP, and the other containing 0.5 mM internal GDP.
Traces were obtained in the absence of GDP (trace a), with 0.5
mM external GDP only (trace b), with 0.5 mM internal GDP only (trace c), and with 0.5 mM GDP
on both sides of the membrane (trace d). Panel B,
K
flux in the presence of laurate. The traces obtained
from PBFI fluorescence were converted to intraliposomal
[K
]
([K
]
) and plotted versus time. K
flux was induced by addition of 0.1
µM valinomycin (arrow) in the presence of 20
µM laurate. In this experiment, valinomycin-mediated
K
flux reflects the limiting flux of
H
, as measured in panel A. The presence or
absence of internal and/or external GDP is designated exactly as for panel A. Panel C, K
flux in the
presence of undecanesulfonate. The experiment was carried out exactly
as described for panel B, except that 20 µM undecanesulfonate was present in the assay medium. The presence or
absence of internal and/or external GDP is designated exactly as for panel A. Media compositions for all three panels are given
under ``Experimental
Procedures.''
An acidification jump can be seen following addition
of laurate when H flux was measured (20-24 s, Fig. 1A). This reflects rapid equilibration of lauric
acid across the membrane. A capacitative K
jump can be
seen following addition of valinomycin when K
flux was
measured (Fig. 1, B and C).
These
experiments establish several key aspects of UCP behavior. (i) The
observed fluxes were electrophoretic, because they required valinomycin
and a K gradient. (ii) The observed fluxes required
addition of 10-100 µM of a long chain FA or
alkylsulfonate. In their absence, observed fluxes were nearly identical
with trace d in each figure (see below). (iii) The observed
fluxes required UCP and were not observed in liposomes lacking UCP (not
shown). (iv) Fluxes were completely inhibited when GDP was present on
both sides of the membrane. Thus, traces from identical experiments in
liposomes lacking UCP were superimposable on the lowest traces of each
panel in Fig. 1. (
)(v) The pattern of GDP-sensitivity
of fluxes induced by 10
M laurate and
10
M undecanesulfonate (Fig. 1) is
entirely similar to that previously observed for flux induced by
10
M Cl
(2) .
Thus, these fluxes were partially inhibited when GDP was present on one
side of the membrane and fully inhibited when GDP was present on both
sides. This reflects the facts that the GDP binding site is accessible
only from one side of the protein (17) and UCP is more or less
randomly inserted into the liposomal membrane(5) .
[vi] K
flux in the presence of valinomycin
is a reliable measure of charge movement through UCP.
Figure 2:
Laurate, but not undecanesulfonate,
activates H efflux from proteoliposomes reconstituted
with UCP. H
flux, determined from changes in
intraliposomal [TES
], is plotted versus concentrations ([Anion]) of undecanesulfonate (
)
and laurate (
). Laurate or undecanesulfonate were added to the
proteoliposomes in assay medium, followed by addition of 0.1 µM valinomycin to initiate H
efflux, as in Fig. 1A. Net fluxes are plotted after subtraction of
flux obtained in the presence of 1 mM external GDP. Nonlinear
regression (solid line) of the dose-response curve yielded K
of 9 µM for laurate. For
undecanesulfonate, the dashed line merely connects the points.
Media compositions were identical for the two anions and are described
under ``Experimental
Procedures.''
As
shown in Fig. 1, B and C, both laurate and
undecanesulfonate catalyzed net charge movement via UCP at comparable
rates. These differential effects on H transport were
confirmed in swelling experiments on intact brown adipose tissue
mitochondria (not shown). The results of these experiments support two
important conclusions. The behavior of undecanesulfonate differs from
that of its close analogue, laurate, and measurements of K
flux under the conditions of Fig. 1C are
reporting influx of the undecanesulfonate anion and not efflux of
H
.
Figure 3:
Kinetics of undecanesulfonate influx and
laurate-induced H efflux in proteoliposomes
reconstituted with UCP. J
refers to
undecanesulfonate influx (
), measured indirectly as K
influx, and laurate-induced H
efflux (
),
measured directly. [Anion] refers to concentrations
of undecanesulfonate and laurate, respectively. The figure contains
Eadie-Hofstee plots for the two anions. Net fluxes are plotted after
subtraction of flux obtained in the presence of 1 mM external
GDP. Except for internal probe, media compositions were identical for
the two anions and are described under ``Experimental
Procedures.'' Linear regressions (solid lines) of the
data yielded a K
of 12 µM and a V
of 37,600 nmol/min mg of protein
for undecanesulfonate and a K
of 8
µM and a V
of 22,000 nmol/min mg of
protein for laurate.
Figure 4:
Laurate and undecanesulfonate inhibit
Cl influx into proteoliposomes reconstituted with
UCP. Percent inhibition of UCP-mediated Cl
influx is
plotted versus [laurate] (
) and
[undecanesulfonate] (
) ([Anion]). Nonlinear
regressions (solid lines) yielded IC
values of 23
and 32 µM for undecanesulfonate and laurate, respectively.
Hill coefficients for both curves are 1. Cl
flux was
determined from SPQ fluorescence as described in Experimental
Procedures.
Figure 5:
Laurate and undecanesulfonate are
competitive inhibitors of UCP-mediated Cl transport.
UCP-mediated Cl
influx (J
) was
measured as described under ``Experimental Procedures.'' J
was measured in varying Cl
concentrations ([Cl
]) without
additions (
) and containing 10 µM laurate (
)
or 50 µM undecanesulfonate (
).
[Cl
] was varied by mixing medium containing
175.5 mM KCl with medium containing 175.5 mM potassium glucuronate. Internal medium was adjusted with
TEA
SO
to be isosmotic with external medium.
Cl
uptake was initiated with 0.1 µM valinomycin. Linear regressions (solid lines) of the
initial rates yielded apparent K
values
for Cl
of 140 mM (
), 164 mM (
), and 332 mM (
). Corresponding V
values (nmol of Cl
/min mg of
protein) were 9490 (
), 8800 (
), and 8530 (
).
Assuming fully competitive inhibition, the K
values calculated for undecanesulfonate and laurate were 37
and 66 µM, respectively.
Figure 6:
Undecanesulfonate is a competitive
inhibitor of laurate-induced H efflux in
proteoliposomes reconstituted with UCP. The dependence of H
efflux on [laurate] is expressed as a double-reciprocal
plot without further addition (
) and in the presence of 100
µM undecanesulfonate (
). J - J
is the difference in flux in the presence (J) and absence (J
) of laurate.
H
efflux was measured as described under
``Experimental Procedures.'' Linear regressions of the data (solid lines) yielded apparent K
values for laurate of 22 and 53 µM in the
absence and presence of 100 µM undecanesulfonate,
respectively. Based on purely competitive inhibition, the K
value for undecanesulfonate was
calculated to be 72 µM.
The traces in Fig. 7demonstrate that lauric acid equilibrates rapidly across the membrane, resulting in the delivery of protons to the intraliposomal space of protein-free liposomes. In contrast, undecanesulfonate additions were without effect on intraliposomal pH. Thus, undecanesulfonic acid is not transported by nonionic diffusion, probably because its concentration nearly vanishes near neutral pH.
Figure 7:
Lauric acid, but not undecanesulfonic
acid, can diffuse across the liposomal membranes. Inverse SPQ
fluorescence (1/F) is plotted versus time in
liposomes lacking UCP and containing internal medium for H transport (see ``Experimental Procedures''). A decrease
in 1/F indicates protonation of TES anion and, hence, delivery
of protons across the liposomal membrane. Arrows indicate
additions of 25 µM sodium laurate (laurate) or 50
µM sodium undecanesulfonate (undecanesulfonate).
How do FAs activate
UCP-mediated proton transport? This is the principal unsolved question
surrounding the molecular basis of UCP mechanism. Winkler and
Klingenberg (9) propose that multiple FAs bind at sites within
the UCP proton channel, thereby providing local acceptor/donor groups
that facilitate H transport (the Buffering Model).
Based on work showing FA-induced H
transport via the
ADP/ATP carrier, Skulachev (10) proposes that UCP transports FA
anions directly, with protons transported via nonionic diffusion of the
protonated FA (the Protonophoretic Model). Our experiments comparing
the behavior of laurate and its close analogue, undecanesulfonate,
provide a plausible means of distinguishing between these models.
Most features of undecanesulfonate behavior are consistent with
either model. Interpreted according to the Buffering Model, the results
imply that undecanesulfonate competes successfully with laurate in
binding to the FA-binding sites within the UCP proton channel, thereby
inhibiting laurate-induced H transport (Fig. 6). Because it is a strong acid, it provides no buffering
in the channel and therefore cannot support UCP-mediated H
transport (Fig. 2). Interpreted according to the
Protonophoretic Model, undecanesulfonate, like other anions, is
transported through the FA anion channel of UCP. It does not support
UCP-mediated H
transport because it is incapable of
nonionic delivery of protons across the bilayer (Fig. 7).
A
crucial distinction between the two models is that FA binding and
transport is stoichiometrically linked to H transport
in the Protonophoretic Model, whereas FA remain bound to the protein in
the Buffering Model and do not participate stoichiometrically in
H
transport. In this respect, our data support the
Protonophoretic Model on both qualitative and quantitative grounds:
undecanesulfonate anion is transported by UCP. We are unaware
of any physicochemical mechanism to explain why laurate anion should
remain bound without also undergoing transport. Furthermore,
undecanesulfonate, as a stand-in for laurate anion, is transported with V
and K
values that are
fully sufficient to account for laurate-induced H
transport via a stoichiometric, protonophoretic mechanism (Table 1). Applying Occam's razor, we infer that FA
catalyze UCP-mediated proton conductance because the FA anion is
transported by UCP and the protonated FA is rapidly transported across
the lipid bilayer by nonionic diffusion (Fig. 8). This proton
cycling mechanism is entirely analogous to uncoupling by weak acid
protonophores. It differs in that a specific protein is required in
order to provide a conductance pathway for back-diffusion of the FA
anion.
Figure 8:
Proposed protonophoretic mechanism of
uncoupling protein. Fatty acid partitions in the membrane and diffuses
laterally to UCP. Fatty acid anion is driven to the center of the
membrane by the electric field along the UCP anion conductance pathway.
The anion flip-flops, and COO reaches the opposite
interface. It then picks up a proton and rapidly flip-flops again,
delivering protons by nonionic diffusion to the other side. This
catalyzed protonophoretic cycle dissipates redox energy and produces
heat. Alkylsulfonates are anions of strong acids and cannot undergo
nonionic diffusion; therefore UCP-mediated alkylsulfonate transport
does not lead to proton transport. For more details, see
``Discussion.''
(i) FA anion partitions in the lipid bilayer with
its head group at the level of the acylglycerol linkages and below the surface of the phospholipid head groups. This shielded
location, driven by the free energy of partitioning of the alkyl chain,
is responsible for the long standing observation that the
pK values of FAs in membranes are 3 to 4 units
higher than their values in solution (22) . Despite high
electrical gradients, there is no significant flux of FA anion, because
the bilayer energy barrier is too high.
(ii) The FA anion diffuses laterally in the bilayer to reach the protein. UCP may contain a weak binding site to concentrate the anion in the conductance pathway. If so, this site must also be partly buried, because kinetic studies show that it is shielded from the bulk aqueous phase(6) .
(iii)
The energy barrier to anion transport is lowered by a weak binding site
located about halfway through the UCP transport pathway. (The existence
of this energy well was deduced from the dependence of UCP-mediated
Cl flux on
(8) ). The anionic head
group is driven to this energy well by the electric field created by
redox-linked proton ejection. Given the preference for hydrophobic
substrates, it seems likely that all or part of the conductance pathway
lies on the outer surface of the protein, at the lipid-protein
interface.
(iv) The anionic carboxyl group moves to the other side of the membrane by a flip-flop mechanism such as occurs during nonionic transmembrane diffusion of protonated FAs(23) . The FA anion then diffuses laterally away from the conductance pathway.
(v) The FA is protonated and rapidly flip-flops again, delivering protons by nonionic diffusion to the mitochondrial matrix and completing the cycle.
It should be emphasized that long
chain FAs that reach the matrix by nonionic diffusion cannot enter the
-oxidation pathway, because the matrix doesn't contain long
chain acyl-CoA synthetase(21) . Instead, acylcarnitine is
transported to the matrix where it is activated by carnitine
acyltransferase II. Since FA rapidly equilibrate across the inner
membrane, it may be useful to view carnitine/acylcarnitine translocase
as being required for channeling of long chain FAs into the
-oxidation pathway.