(Received for publication, July 18, 1995; and in revised form, September 7, 1995)
From the
The cross-linking reagent copper-o-phenanthroline
complex (Cu(OP)) specifically caused a decrease in the
amount of the 30-kDa ADP/ATP carrier in bovine submitochondrial
particles associated predominantly with formation of a 60-kDa protein
consisting of a cross-linked dimer of the carrier. However,
Cu(OP)
had no effect on mitochondria. The transport of ADP
via the carrier through submitochondrial particle membranes was found
to be inhibited in parallel with the progress of intermolecular
cross-linking. Analysis of the cross-linked site showed that a
disulfide bridge was formed only between two Cys
residues
in a pair of the first loops facing the matrix space. The transport
inhibitor bongkrekic acid, which locks the m-state conformation of the
carrier, had no effect on disulfide bridge formation catalyzed by
Cu(OP)
, but carboxyatractyloside, which locks the c-state
conformation by acting from the cytosolic side, completely inhibited
the cross-linking. These results show that the ADP/ATP carrier
functions as a dimer form, and a pair of the first loops protrudes into
the matrix space in the m-state, but possibly intrudes into the
membrane in the c-state. Thus, it is suggested that a pair of the first
loops acts as a gate and that its opening and closing are regulated by
their translocation.
The 30-kDa ADP/ATP carrier present in the inner mitochondrial membrane mediates the transport of ADP and ATP from both the cytosolic and matrix sides of the mitochondrial inner membrane, and its structural properties including its primary structures in preparations from various sources have been well studied(1, 2, 3) . The carrier consists of three repeated homologous domains(4, 5) , and a six-transmembrane model is proposed(2, 3, 5, 6) . In this model, each repeat consists of a pair of transmembrane segments linked by a hydrophilic matrix-facing loop. These loops are referred to as M1, M2, and M3. The functional unit of the carrier is considered to be a dimer form(7, 8, 9) , although other possibilities, such as a tetrameric (10) or monomeric (11) carrier as the functional unit, have been proposed.
Chemical modifications of certain amino acid residues provide useful
information about the structure of proteins. From the discrete
reactivities of the cysteine residues in the ADP/ATP carrier with the
maleimide SH reagents NEM ()and eosin 5-maleimide, we
concluded that of the four cysteine residues, only Cys
is
located in the membrane segment, while Cys
,
Cys
, and Cys
are in the three matrix-facing
loops M1, M2, and M3, respectively, being consistent with the
six-transmembrane model(12, 13) . We also found that
loop M1 is exposed to the matrix space, loop M2 intrudes into the
membrane, and loop M3 is located deep in the membrane. Moreover, we
concluded that loops M1 and M2 act as gates in the transport of ADP and
ATP. The role of loop M3 is not yet clear. Based on these results, we
proposed a model of adenine nucleotide transport involving changes in
the locations of pairs of M1 loops and M2 loops in the dimer form of
the carrier(13) .
Cu(OP), a well known
cross-linking reagent, catalyzes the formation of disulfide bonds
between SH groups(14) , and it has been used to determine the
topologies and oligomeric states of membrane
proteins(15, 16, 17) . Zimmer and co-workers (18, 19) examined the effects of cross-linking
reagents including Cu(OP)
on an oligomycin-sensitive ATPase
preparation from bovine heart mitochondria and found that it modified a
31-kDa protein significantly. Later, Joshi and Torok reported that an
ADP/ATP carrier contaminating the preparation of ATPase was the protein
modified by Cu(OP)
and that Cu(OP)
caused
intermolecular and intramolecular disulfide bridges in electron
transport particles from bovine heart mitochondria(20) , but it
seemed to induce only intramolecular cross-linking of the carrier
present in the preparation of H
-ATPase(21) .
They also suggested that Cys
and Cys
were
associated with the intramolecular cross-linking.
As we proposed
previously that the ADP/ATP carrier is responsible for the uncoupling
induced by Cu(OP)(22) , these results prompted us
to study which cysteine residues are responsible for the intermolecular
cross-linkage and which of the intermolecular or intramolecular
disulfide bond(s) is formed predominantly in the membrane-bound
carrier. Therefore, we examined the effects of Cu(OP)
on
bovine heart submitochondrial particles under various conditions. Based
on our present results, we discuss changes in configuration of the
loops in the ADP/ATP carrier in relation to its transport function.
Particles (10 mg of
protein) that had been incubated with or without 100 µM Cu(OP) for 20 min at 0 °C were treated with 5
mM EDTA and 5 mM NEM to terminate the reaction and to
alkylate free SH groups, respectively. Then they were solubilized in a
solution of 5% Triton X-100, 0.5 M NaCl, 0.5 mM EDTA,
and 10 mM MOPS (pH 7.2) for 10 min at O °C. The
solubilized sample was passed through a hydroxylapatite column, and the
flow-through fraction was treated with 5 volumes of acetone at
-20 °C overnight. The precipitate was collected by
centrifugation, washed with diethyl ether, and dried under vacuum. The
dried sample was dissolved in 6 M guanidine HCl containing 2
mM EDTA, 1 mM NEM, and 100 mM MOPS (pH 6.8)
and incubated for 60 min at 25 °C to alkylate the free SH groups
with NEM completely. Then (AAC)
or AAC was isolated by gel
filtration on a G4000SW
column (0.78
30 cm; Tosoh
Co., Tokyo) with 63% acetonitrile containing 0.05% trifluoroacetic acid
at a flow rate of 0.5 ml/min. The fractions containing (AAC)
(5 nmol) or AAC (10 nmol) were concentrated in a SpeedVac
concentrator and denatured with 10 volumes of 6 M guanidine
HCl solution containing 1 mM EDTA and 0.5 M Tris-HCl
buffer (pH 8.5). Disulfide bonds in proteins were reduced by incubation
with 2 µmol of DTT for 2 h at 37 °C under a stream of nitrogen,
and the resulting reduced cysteine residues were carboxamidomethylated
with 4.2 µmol of iodoacetamide for 30 min at 25 °C in the dark.
These preparations are referred to as RC-(AAC)
and RC-AAC,
respectively. The solutions were acidified with trifluoroacetic acid
and subjected to HPLC on a G4000SW
column with 63%
acetonitrile containing 0.05% trifluoroacetic acid. Peak fractions
containing RC-(AAC)
or RC-AAC were concentrated to 50
µl and diluted four times with 200 mM Tris-HCl buffer (pH
8.0), and samples were digested with lysylendopeptidase (2%, w/w) at 37
°C for 24 h. After addition of 5 volumes of 7 M guanidine
HCl, the digested peptides were separated by reversed-phase HPLC on a
TSKgel ODS-120T column (0.46
15 cm; Tosoh Co.) using a linear
gradient of 12-52% acetonitrile containing 0.05% trifluoroacetic
acid in 80 min at a flow rate of 1.0 ml/min. The elution was monitored
by the absorbance at 210 nm.
As a reference, the ADP/ATP carrier was
isolated from the particles without Cu(OP) treatment by the
same procedure as described above. The isolated carrier was denatured
with guanidine HCl and reduced with DTT. Then all its cysteine residues
were either alkylated with NEM or carboxamidomethylated with
iodoacetamide. The carriers thus obtained are referred to as RNEM and
RCAM, respectively.
Figure 1:
Effect of concentration of Cu(OP) on membrane proteins in bovine heart submitochondrial particles.
Bovine heart submitochondrial particles (4 mg of protein/ml) in medium
consisting of 250 mM sucrose and 10 mM Tris-HCl
buffer (pH 7.4) were incubated with Cu(OP)
for 10 min at 0
°C, and the oxidative reaction was terminated with 5 mM EDTA and 5 mM NEM. After 10 min, samples of particle
suspension containing 10 µg of protein were subjected to SDS-PAGE
under nonreducing conditions, and proteins were stained with Coomassie
Blue.
Figure 2:
Immunostaining of the ADP/ATP carrier in
submitochondrial particles treated with Cu(OP). After
SDS-PAGE as shown in Fig. 1, the proteins in the gel were
transferred electrophoretically to a polyvinylidene difluoride membrane
and incubated with rabbit anti-ADP/ATP carrier serum (1:1000 dilution).
The membrane was further incubated with peroxidase-conjugated goat
anti-rabbit IgG (1:2000 dilution) and then developed with
3-amino-9-ethylcarbazole (0.17 mg/ml) and 0.015% hydrogen peroxide for
10 min (A) or 60 min (B).
Figure 4:
Effects of various compounds on
cross-linking of the ADP/ATP carrier catalyzed by Cu(OP).
The effect of 2 mM NEM was examined by its incubation with
bovine heart submitochondrial particles (20 mg of protein/ml) for 10
min at 0 °C in medium consisting of 250 mM sucrose and 10
mM Tris-HCl buffer (pH 7.4) in a similar way to that described
previously(12) . For examination of the effects of
solubilization with detergents, the particles (8 mg of protein/ml) were
treated with either 1% SDS or 3% Triton X-100 for 5 min at 0 °C in
medium consisting of 150 mM Na
SO
and
20 mM MOPS (pH 7.4), which is a commonly used medium for
reconstitution in experiments on the ADP/ATP carrier(25) . All
samples (4 mg of protein/ml) were treated with 100 µM Cu(OP)
as described for Fig. 1and then
subjected to SDS-PAGE under nonreducing conditions (left) or
under reducing conditions with 4% 2-mercaptoethanol (2ME) (right). SMP indicates submitochondrial particles
without any treatment, and None indicates submitochondrial
particles treated only with
Cu(OP)
.
Fig. 3shows the changes in the
amounts of AAC, (AAC), and AAC` determined from the
intensities of the immunostained bands shown in Fig. 2as
functions of the Cu(OP)
concentration. With an increase in
the concentration of Cu(OP)
, the amount of AAC decreased
rapidly up to 100 µM Cu(OP)
, and then more
gradually. With 400 µM Cu(OP)
,
90% of the
carrier was modified. With a decrease in the amount of AAC, on
treatment with Cu(OP)
up to 100 µM, the level
of (AAC)
increased
60% and then decreased gradually
due to formation of aggregates, which remained at the origin of the gel (cf.Fig. 1). In contrast, formation of AAC` increased
slightly but steadily with an increase in the Cu(OP)
concentration, attaining a plateau level of
10% of the
original amount of AAC at 400 µM Cu(OP)
.
Similar changes were observed in the Coomassie Blue staining
intensities of the AAC and (AAC)
bands shown in Fig. 1. However, their levels were not superimposable on those
of the corresponding immunostained bands. Results with AAC are shown in Fig. 3, the intensity level being consistently higher than that
of the immunostained band due to overlapping of the AAC band with some
other protein bands of
30 kDa.
Figure 3:
Progressions of intermolecular and
intramolecular disulfide bond formation of the ADP/ATP carrier on
treatment with Cu(OP). The intensities of immunostained
bands of AAC, (AAC)
, and AAC` in Fig. 2were
determined by densitometry at 510 nm. The immunostaining intensities
are shown relative to that of AAC in submitochondrial particles without
Cu(OP)
treatment. The broken line represents the
change in Coomassie Blue staining intensity of the AAC band shown in Fig. 1.
These results showed that
Cu(OP) caused formation of mainly (AAC)
and
then higher aggregates. During these changes, transformation of AAC to
AAC` also took place, but to a much lesser extent. At higher
temperatures, such as 25 °C, and at alkaline pH values, such as pH
8, the effect of Cu(OP)
was more pronounced, and formation
of higher aggregates proceeded so rapidly that the 60-kDa band was
hardly observable (data not shown). It is noteworthy that under these
conditions, formation of intramolecular cross-linked AAC` became
significant.
As the formation of higher aggregates and
intramolecular cross-linking were very low at Cu(OP) concentrations of <100 µM at 0 °C, these mild
conditions were suitable for quantitative analyses of the effect of
Cu(OP)
. As shown in Fig. 4, the 60-kDa band of
(AAC)
formed by treatment of the particles with 100
µM Cu(OP)
for 10 min at 0 °C completely
disappeared on further treatment with SH-reducing reagents such as
2-mercaptoethanol, and instead, the 30-kDa band of AAC was restored to
the same level as in the untreated particles. Pretreatment of particles
with NEM for 10 min completely inhibited the formation of
(AAC)
. These results indicate that Cu(OP)
oxidizes SH groups of the carrier, resulting predominantly in the
formation of intermolecular disulfide bridges.
When submitochondrial
particles were solubilized with 3% Triton X-100 and then treated with
Cu(OP), no 60-kDa (AAC)
band was observed, and
the amount of 28-kDa AAC` increased significantly in association with
the disappearance of the 30-kDa band (Fig. 4). Moreover,
treatment of the ADP/ATP carrier in submitochondrial particles first
with 1% SDS and then with Cu(OP)
caused the appearance of a
band at
29 kDa distinct from that at 28 kDa formed on treatment
with Triton X-100. As these two bands were reversed to the band of 30
kDa by treatment with 2-mercaptoethanol (Fig. 4), they were
concluded to be due to intramolecular cross-linking of cysteine
residues catalyzed by Cu(OP)
and to have slightly different
conformations from 30-kDa AAC, resulting in their higher
electrophoretic mobilities on SDS-PAGE.
Next, we examined the time
course of the cross-linking of the carrier by Cu(OP) and
the effect of this cross-linking on ADP transport. For this, we
incubated submitochondrial particles with 100 µM Cu(OP)
at 0 °C for various periods, terminating
the oxidation of cysteine residues by addition of EDTA. Then we
subjected part of the preparation to SDS-PAGE and used another part for
determination of the ADP transport activity. As shown in Fig. 5A, within the first 10 min, the Coomassie Blue
staining intensity of the carrier at 30 kDa decreased rapidly with an
increase in that of (AAC)
. Subsequently, the rate of
decrease in intensity of the 30-kDa band became slower, while that of
the 60-kDa band attained a plateau level due to formation of higher
aggregates. The ADP transport activity of the ADP/ATP carrier was
inhibited by Cu(OP)
in accordance with a decrease in the
amount of the 30-kDa carrier (Fig. 5B). The
time-dependent decrease in intensity of the band of AAC stained with
Coomassie Blue was qualitatively, but not quantitatively, similar to
that of ADP transport inhibition because the AAC band overlapped those
of other proteins of
30 kDa, as described above (cf.Fig. 3). As the relative transport inhibition (
26%)
after treatment with 100 µM Cu(OP)
for 10 min
at 0 °C was consistent with the relative amount of AAC determined
by the immunostaining (
20%) shown in Fig. 3under the same
incubation conditions, cross-linking of the carrier was concluded to be
associated with inhibition of transport. Incubation of the mitochondria
with 100 µM Cu(OP)
for up to 60 min resulted
in no appreciable change in the 30-kDa band (data not shown), showing
that Cu(OP)
, which oxidizes SH groups on the membrane
surface(17, 24) , interacted only with SH groups on
the matrix side, not with those on the cytosolic side.
Figure 5:
Time course of the effect of Cu(OP) on the ADP/ATP carrier and its ADP transport activity in bovine
heart submitochondrial particles. Submitochondrial particles were
treated with 100 µM Cu(OP)
for various periods
at 0 °C under the same conditions as described for Fig. 1,
and reactions were terminated by addition of 5 mM EDTA. A
portion of each sample was subjected to SDS-PAGE, and the Coomassie
Blue staining intensities of the bands were measured at 560 nm (A). Another portion was diluted to 2 mg of protein/ml with
medium consisting of 250 mM sucrose, 1 µg of oligomycin/mg
of protein, 0.1 mM EDTA, and 10 mM Tris-HCl buffer
(pH 7.2) at 0 °C and incubated for 5 min at the same temperature.
Then [
H]ADP was added at a final concentration of
20 µM, and after 10 s, transport of ADP was terminated
with 20 µM BKA, and the amount of
[
H]ADP transported was determined (B).
Results are means ± S.D. for three separate
runs.
Figure 6:
Isolation of the ADP/ATP carrier and its
intermolecular cross-linked dimer formed by treatment with Cu(OP) from bovine heart submitochondrial particles. Submitochondrial
particles treated with 100 µM Cu(OP)
for 20
min at O °C and those without treatment were solubilized with 5%
Triton X-100 in 0.5 M NaCl, 0.5 mM EDTA, and 10
mM MOPS (pH 7.2), and the solubilized samples were passed
through a hydroxylapatite gel column. Proteins in the flow-through
fractions were denatured with 6 M guanidine HCl, and their
free SH groups were completely alkylated with 1 mM NEM. The
protein samples were eluted from a G4000SW
column (0.78
30 cm) with 63% acetonitrile containing 0.05% trifluoroacetic
acid at a flow rate of 0.5 ml/min, and fractions of the eluate were
collected at 1-min intervals. Elution was monitored at 280 nm. The
major peaks 1 and 2 were observed with protein
samples derived from submitochondrial particles with and without
Cu(OP)
treatment, respectively (A). The proteins
in peaks 1 and 2 gave single bands on nonreducing SDS-PAGE (B), and these peaks were identified as (AAC)
and
AAC, respectively.
In addition, we prepared ADP/ATP carrier in which all the
cysteine residues were alkylated with NEM or carboxamidomethylated with
iodoacetamide. These carrier preparations are referred to as RNEM and
RCAM, respectively. RNEM and RCAM were digested with
lysylendopeptidase, and their peptide fragments were separated by
reversed-phase HPLC. As shown in Fig. 7(A and B), peaks due to the peptides
Gly-Lys
,
Gly
-Lys
,
Gln
-Lys
, and
Gln
-Lys
, containing
Cys
, Cys
, Cys
, and
Cys
, respectively, were observed. We identified these
four peaks of peptides containing cysteine residues by amino acid
sequence analysis as described previously(12) . The peptides
alkylated by NEM are referred to as N159, N256, N56, and N128,
respectively, and those carboxamidomethylated as C159, C256, C56, and
C128, respectively, according to the cysteine positions in these
peptide fragments.
Figure 7:
Reversed-phase HPLC profiles of peptide
fragments from the ADP/ATP carrier and its intermolecular cross-linked
dimer. The isolated ADP/ATP carrier was denatured with 6 M guanidine HCl, reduced with 6.7 µmol of DTT/mg of protein, and
then alkylated with 14 µmol of NEM/mg of protein (referred to as
RNEM) or carboxamidomethylated with 14 µmol of iodoacetamide/mg of
protein (referred to as RCAM). RNEM and RCAM derived from the isolated
carrier without Cu(OP) treatment were used as references.
Fractions of AAC and (AAC)
obtained by chromatography on
G4000SW
as shown in Fig. 6were denatured with
guanidine HCl and reduced with DTT, and the free SH groups that
appeared were carboxamidomethylated. Carboxamidomethylated AAC and
(AAC)
are referred to as RC-AAC and RC-(AAC)
,
respectively. All samples were digested with lysylendopeptidase, and
their peptide fragments were separated by reversed-phase HPLC with a
linear gradient of 0-12% acetonitrile containing 0.05%
trifluoroacetic acid for 5 min and 12-52% for 80 min at a flow
rate of 1 ml/min. Elution was monitored at 210 nm. N56, N128, N159, and N256 are peaks of peptide
fragments containing Cys
, Cys
,
Cys
, and Cys
, respectively, alkylated with
NEM, and C56, C128, C159, and C256 are those of the respective carboxamidomethylated
derivatives.
For identification of the cysteine residues
involved in formation of intermolecular disulfide bonds catalyzed by
Cu(OP), (AAC)
was isolated, denatured, and
alkylated with NEM under nonreducing conditions. Then the preparation
was treated with DTT to reduce all the disulfide bonds, and the
resulting free SH residues were carboxamidomethylated. As all the free
cysteine residues in denatured (AAC)
had been alkylated
with NEM, the cysteine residues that were carboxamidomethylated should
be involved in the intermolecular disulfide bonds. We also treated
isolated AAC with iodoacetamide under reducing conditions after
denaturation and alkylation by NEM following the same procedure. These
carboxamidomethylated AAC and (AAC)
preparations are
referred to as RC-AAC and RC-(AAC)
, respectively. They were
digested with lysylendopeptidase and then subjected to reversed-phase
HPLC.
The elution profile of peptide fragments from RC-AAC showed
four peaks due to N159, N256, N56, and N128, but no peak due to
peptides containing carboxamidomethylated cysteine residue was observed (Fig. 7C), indicating that all the cysteine residues in
AAC had been alkylated with NEM before reduction and
carboxamidomethylation and that no disulfide bridge exists in intact
ADP/ATP carrier. In contrast, the elution profile of RC-(AAC) showed a peak due to C56 besides those due to N159, N256, and
N128, which were almost the same heights as those of RCAM and RNEM. No
peak due to N56 or to C159, C256, or C128 was detected (Fig. 7D). These results clearly show that Cys
is responsible for disulfide bridge formation catalyzed by
Cu(OP)
and that no other cysteine residues form
cross-linkages. We concluded that a disulfide bond was formed
specifically between two Cys
residues in a pair of M1
loops of the dimeric ADP/ATP carrier.
Figure 8:
Effects of specific transport inhibitors
on cross-linking of the ADP/ATP carrier. Submitochondrial particles
were preincubated with or without various concentrations of CATR or BKA
for 10 min at 0 °C and then treated with 50 µM Cu(OP) for 10 min at 0 °C. Submitochondrial
particles (SMP) without treatment with Cu(OP)
were
also prepared. Samples (10 µg of protein) were subjected to
SDS-PAGE, and the Coomassie Blue staining intensity of the cross-linked
dimer (AAC)
was determined. A, nonreducing
SDS-PAGE of proteins in submitochondrial particles after incubation
with 40 µM BKA and CATR. CATR was added from the cytosolic
side (CATR(c)) before preparation of submitochondrial
particles or from the matrix side (CATR(m)). B,
Coomassie Blue staining intensities of (AAC)
at various
concentrations of BKA and CATR.
Next, the effects of 50 µM Cu(OP) on the
disulfide bond formation of the carrier in submitochondrial particles,
which had been pretreated with these inhibitors at 40 µM,
were examined at various incubation times with Cu(OP)
at 0
°C. The time courses of changes in Coomassie Blue staining
intensities of AAC and (AAC)
are shown in Fig. 9.
The decrease in the amount of AAC and the increase in the amount of
(AAC)
were more gradual than those on treatment with 100
µM Cu(OP)
(cf.Fig. 5). As
expected, BKA added from the matrix side had no effect on the formation
of (AAC)
, but CATR added from the cytosolic side completely
inhibited loss of AAC and formation of (AAC)
even on long
incubation (60 min) with Cu(OP)
. As BKA from the matrix
side and CATR from the cytosolic side fix the m-state and c-state
conformations of the carrier, respectively(6) , we conclude
that the m-state, but not the c-state, conformation of the carrier
favors formation of the disulfide bridge catalyzed by
Cu(OP)
.
Figure 9:
Time courses of formation of the
intermolecular cross-linked ADP/ATP carrier on treatment with
Cu(OP) in the presence and absence of BKA and CATR.
Submitochondrial particles pretreated with 40 µM BKA or
CATR were incubated with 50 µM Cu(OP)
for the
indicated times at 0 °C and then subjected to SDS-PAGE. Coomassie
Blue staining intensities of the carrier (AAC) and its cross-linked
dimer ((AAC)
) were determined. Experimental conditions were
as described for Fig. 8.
The cross-linking reagent Cu(OP) has been
reported to cause specific intermolecular and intramolecular
cross-linking of the ADP/ATP carrier in submitochondrial particles (20) and apparently to catalyze formation of only
intramolecular cross-links associated with Cys
and
Cys
in the carrier contaminating a preparation of
H
-ATPase(21) . Therefore, the susceptibility
of the membrane-bound ADP/ATP carrier to Cu(OP)
may be
different from that in the isolated preparation. In this study, we
confirmed that Cu(OP)
caused cross-linking of the carrier
in bovine heart submitochondrial particles, but did not affect the
carrier in mitochondria. Our results show that four cysteine residues
(Cys
, Cys
, Cys
, and
Cys
) in the carrier are not exposed to the cytosolic
side, as we reported previously(12, 13) . Formation of
intermolecular disulfide bridges took place predominantly under mild
oxidation conditions with Cu(OP)
, whereas formation of the
intramolecular bridge occurred under rather strong oxidation
conditions.
It is noteworthy that the carrier solubilized by Triton
X-100 and that denatured by SDS did not form the dimer
(AAC) even under mild conditions, but instead formed
intramolecular cross-links with different conformations as reflected by
different electrophoretic mobilities on SDS-PAGE. Therefore, even the
carrier solubilized by the ``mild'' detergent Triton X-100,
which has been commonly used to isolate the ADP/ATP carrier for
reconstitution experiments(25) , may have a different
conformation from that of the membrane-bound carrier. This is
consistent with the predominant formation of intramolecular cross-links
of the ADP/ATP carrier in isolated preparations of
H
-ATPase(21) . Our results suggest that under
mild oxidation conditions, Cu(OP)
could be a useful probe
to monitor the integrity of the ADP/ATP carrier in the membrane.
Interestingly, only Cys in loop M1 was associated with
intermolecular cross-linking, indicating that this loop is exposed to
the matrix space, as we suggested previously(12, 13) .
The Cys
residues are not always located very close
together because they did not form disulfide bridges in
``intact'' submitochondrial particles in the absence of
Cu(OP)
. Probably, they fluctuate in position in the
membrane as observed with the C-terminal peptide of uncoupling protein,
a member of the mitochondrial solute carrier family like the ADP/ATP
carrier(17) . The present results also confirm that the dimer
form of the ADP/ATP carrier is its functional unit (7, 8, 9) and that two carrier molecules are
located in the membrane facing each other in the same orientation. As
intermolecular cross-linking inhibited ADP transport via the ADP/ATP
carrier, a pair of M1 loops may act as a gate in adenine nucleotide
transport. In contrast, intermolecular cross-linking of cysteine
residues in the C-terminal region of uncoupling protein did not inhibit
H
transport, showing that this peptide chain does not
act as a gate(17) .
It is known that the ADP/ATP carrier has
two conformational states, the c- and m-states, and that adenine
nucleotides are transported due to interconversion of these two
states(6, 13) . The transport inhibitors CATR, acting
from the cytosolic side, and BKA, acting from the matrix side, fix the
c-state and m-state conformations,
respectively(2, 3) . In the m-state conformation fixed
by BKA, the carrier easily formed intermolecular cross-links on
treatment with Cu(OP) (in a similar manner to that without
BKA), but in the c-state conformation fixed by CATR, cross-linking was
completely inhibited, indicating that in the m-state, a pair of M1
loops is located in such a way that it protrudes from the membrane,
whereas in the c-state, it possibly intrudes into the transport path
consisting of 12 transmembrane segments of the dimeric carrier, as
depicted schematically in Fig. 10. Therefore, translocation of
M1 loops from the matrix side to the membrane segment should be
associated with the opening and closing of the gate consisting of a
pair of M1 loops, namely, the gate is open in the m-state and closed in
the c-state. The findings that the loops facing the matrix side of the
ADP/ATP carrier are susceptible to site-specific proteases (26, 27) and to the SH reagents NEM (28) and
eosin 5-maleimide (13) only in the m-state, but not in the
c-state, support this model of open-closed transconversion of a pair of
M1 loops. In addition, these changes in the location of loop M1 should
be responsible for the large conformation change of the carrier between
the m- and c-states monitored by its intrinsic fluorescence (29) and by extrinsic fluorescent derivatives of ADP and ATP
bound to the carrier (30) .
Figure 10: Schematic representation of the locations of loops of the dimer form of the ADP/ATP carrier in two conformational states. The model depicts 12 cylindrical transmembrane segments connected with hydrophilic loops in the dimer form of the carrier according to the six-transmembrane model(2, 3, 5, 6, 12) . In the m-state conformation, a pair of M1 loops (between transmembrane segments 1 and 2) protrudes into the matrix space, while loop M2 (between transmembrane segments 3 and 4) and loop M3 (between transmembrane segments 5 and 6) intrude into the transport path. In the c-state conformation, a pair of M1 loops is translocated to the transport path, and loops M2 and M3 remain in the membrane. Therefore, the gate for transport consisting of a pair of M1 loops is open in the m-state, but closed in the c-state.
In a previous paper(13) , we proposed a transport model of ADP and ATP via the ADP/ATP carrier based on the discrete labelings of the four cysteine residues of the carrier with the maleimide SH reagent eosin 5-maleimide in the presence and absence of BKA and CATR. According to this model, the binding of the transport substrates ADP and ATP from the matrix side to their primary binding site, loop M2, causes opening of the gate formed by a pair of these loops associated with the closing of the gate consisting of a pair of M1 loops. Transport of adenine nucleotides from the matrix side to the cytosolic side is accomplished by the opening and closing of these gates, and the carrier is thereby converted from the m-state to the c-state conformation. Similarly, the binding of adenine nucleotides from the cytosolic side results in the closing of the gate consisting of a pair of M2 loops associated with the opening of the gate consisting of a pair of M1 loops. In this study, we showed that the gate consisting of a pair of M1 loops is open in the m-state conformation and closed in the c-state conformation by binding of BKA and CATR, respectively, to their primary binding site, loop M2, which is consistent with the above model.
Recently, the translocation of the loop segment of the colicin Ia channel has been reported to be associated with its transport function (31) . Based on this result, Simon (32) proposed that swinging of the loop(s) regulates the transport activity of this channel. Therefore, it is possible that cooperative swinging of pairs of loops of the ADP/ATP carrier is directly associated with the opening/closing of the gates, and adenine nucleotide transport is achieved by these changes. Further study to examine this possibility is under way.