©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Translocation of Loops Regulates Transport Activity of Mitochondrial ADP/ATP Carrier Deduced from Formation of a Specific Intermolecular Disulfide Bridge Catalyzed by Copper-o-Phenanthroline (*)

(Received for publication, July 18, 1995; and in revised form, September 7, 1995)

Eiji Majima (1) (2) Kazuro Ikawa (1) Masashi Takeda (1) Mitsuru Hashimoto (1) Yasuo Shinohara (1) Hiroshi Terada (1)(§)

From the  (1)Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770 and the (2)APRO Life Science Institute, Kurosaki, Naruto 772, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The cross-linking reagent copper-o-phenanthroline complex (Cu(OP)(2)) 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)(2) 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)(2), 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.


INTRODUCTION

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 (^1)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)(2), 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)(2) 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)(2) and that Cu(OP)(2) 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)(2)(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)(2) 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.


MATERIALS AND METHODS

Reagents

NEM was purchased from Nacalai Tesque (Kyoto, Japan). o-Phenanthroline, CuSO(4), and lysylendopeptidase were from Wako Pure Chemical Industries (Osaka, Japan); CATR was from Sigma; and hydroxylapatite and AG 1-X8 were from Bio-Rad. Reagents for HPLC were obtained from Kanto Chemical Co. (Tokyo). BKA and rabbit anti-ADP/ATP carrier serum were gifts from Prof. Duine (Delft University of Technology) and Prof. Klingenberg (University of Munich), respectively.

Preparation of Bovine Heart Submitochondrial Particles

Submitochondrial particles containing 5 mM ATP were prepared from bovine heart mitochondria as described previously(12) , and CATR-preloaded submitochondrial particles were prepared in a similar way from mitochondria that had been incubated with 4 nmol of CATR/mg of protein at 25 °C for 10 min. Amounts of protein in mitochondria and submitochondrial particles were determined with a bicinchoninic acid protein assay kit (Pierce) in the presence of 1% SDS using bovine serum albumin as a standard.

Cross-linking of the ADP/ATP Carrier by Cu(OP)(2)

Cu(OP)(2) was prepared just before use by mixing CuSO(4) with o-phenanthroline in a molar ratio of 1:2 in a solution of 250 mM sucrose and 10 mM Tris-HCl buffer (pH 7.4), and its concentration was expressed in terms of Cu. A solution of Cu(OP)(2) was mixed with an equal volume of submitochondrial particle suspension (8 mg of protein/ml) in the same medium at 0 °C for various periods, and the reaction was terminated by addition of EDTA and NEM, each at a final concentration of 5 mM. For assay by SDS-PAGE, the samples of particles after treatment with Cu(OP)(2) were mixed with an equal volume of 2% SDS in 50 mM Tris-HCl buffer (pH 6.8) containing 20% glycerol and incubated at 25 °C for 30 min. Electrophoresis of 10-µg protein samples was carried out according to Laemmli (23) on 12% polyacrylamide gel. The gel was stained with Coomassie Blue R-250, and the staining intensity of bands was measured at 560 nm in a Shimadzu Chromatoscanner (Model CS-9000). The effects of test compounds on the cross-linking of the ADP/ATP carrier were examined by their preincubation with submitochondrial particles at 0 °C for 10 min.

Immunostaining of the ADP/ATP Carrier

Proteins in the gel after electrophoresis were transferred electrophoretically to a polyvinylidene difluoride membrane (Bio-Rad) in a semidry blotter (Sartorius, Göttingen, Germany) with anode buffer (No. 1) consisting of 0.3 M Tris and 20% methanol; anode buffer (No. 2) consisting of 25 mM Tris and 20% methanol; and cathode buffer consisting of 25 mM Tris, 40 mM 6-amino-n-caproic acid, and 20% methanol in the presence of 0.02% SDS for 1 h at 3 mA/cm^2. Unoccupied free binding sites on the membrane were blocked by incubation with 10% bovine serum albumin for 1 h at room temperature. Then the membrane was incubated overnight with TBS (25 mM Tris-HCl and 0.15 M NaCl (pH 7.4)) containing 0.1% bovine serum albumin and rabbit anti-ADP/ATP carrier serum (1:1000 dilution) at 4 °C. After washing three times with TBS, the membrane was incubated with TBS containing 0.1% bovine serum albumin and peroxidase-conjugated goat anti-rabbit IgG (1:2000 dilution; Cappel) for 1 h at room temperature, washed three times with TBS, and then incubated with 3-amino-9-ethylcarbazole (0.17 mg/ml) and hydrogen peroxide (0.015%) in TBS for 10-60 min at room temperature. Color development was terminated by rinsing the membrane with distilled water and then drying it.

Assay of ADP Transport Activity

Submitochondrial particles with or without Cu(OP)(2) treatment were suspended at 2 mg of protein/ml in a solution of 250 mM sucrose, 1 µg of oligomycin/mg of protein, 0.1 mM EDTA, and 10 mM Tris-HCl buffer (pH 7.2). Then [^3H]ADP (specific radioactivity, 185 kBq/µmol) was added at a final concentration of 20 µM, and the mixture was incubated for 10 s at 0 °C. Transport was stopped by addition of BKA at a final concentration of 20 µM. Extraparticular ADP was removed by passing the suspension of particles through a column of AG 1-X8. Fractions containing the particles were pooled, and incorporated ADP was determined from the radioactivity(12) .

Determination of Cross-linking Sites

To identify the cross-linked cysteine residues of the ADP/ATP carrier, we isolated the intermolecular cross-linked 60-kDa carrier and the native carrier (30 kDa), referred to as (AAC)(2) and AAC, respectively, from submitochondrial particles with or without Cu(OP)(2) treatment by a method reported previously (12) with slight modifications. The isolated proteins, which had been treated with NEM, were reduced with DTT and alkylated with iodoacetamide. By these treatments, the remaining free SH groups were modified by NEM, and the SH groups responsible for disulfide bonds were carboxamidomethylated. Details of the procedure were as follows.

Particles (10 mg of protein) that had been incubated with or without 100 µM Cu(OP)(2) 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)(2) or AAC was isolated by gel filtration on a G4000SW column (0.78 times 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)(2) (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)(2) 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)(2) 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 times 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)(2) 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.


RESULTS

Cross-linking of the ADP/ATP Carrier by Cu(OP)(2)

First, we examined the effect of Cu(OP)(2) oxidation on the SH groups of the ADP/ATP carrier. For this, submitochondrial particles were incubated with various concentrations of Cu(OP)(2) at 0 °C for 10 min; the reaction was terminated by addition of EDTA to trap free and complexed Cu; and then NEM was added to modify the remaining free cysteine residues. The particles were solubilized with SDS; the solution was subjected to SDS-PAGE; and the Coomassie Blue staining intensities of the bands on the gel were determined. As shown in Fig. 1, the staining intensity of a band at 30 kDa, which was identified as that of the ADP/ATP carrier by amino acid sequence analysis(12) , specifically decreased with an increase in the Cu(OP)(2) concentration, and concomitantly, a 60-kDa band appeared. Both these bands reacted with antibody against the ADP/ATP carrier (Fig. 2A). The 60-kDa band changed back to the 30-kDa band on treatment with 2-mercaptoethanol, as described below (cf.Fig. 4). From these results, the 60-kDa protein was concluded to be formed by intermolecular cross-linking of the ADP/ATP carrier. After treatment with >50 µM Cu(OP)(2), we detected a faint immunostained band at 28 kDa on longer color development of the blotted membrane (Fig. 2B). As the 28-kDa protein in this band changed to the 30-kDa protein on treatment with 2-mercaptoethanol, it was presumably an intramolecular cross-linked carrier, as reported previously(20) . Hereafter, we refer to the 30-kDa ADP/ATP carrier, the intermolecular cross-linked 60-kDa carrier, and the intramolecular cross-linked 28-kDa carrier as AAC, (AAC)(2) and AAC`, respectively.


Figure 1: Effect of concentration of Cu(OP)(2) 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)(2) 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)(2). 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)(2). 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(2)SO(4) 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)(2) 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)(2).



Fig. 3shows the changes in the amounts of AAC, (AAC)(2), and AAC` determined from the intensities of the immunostained bands shown in Fig. 2as functions of the Cu(OP)(2) concentration. With an increase in the concentration of Cu(OP)(2), the amount of AAC decreased rapidly up to 100 µM Cu(OP)(2), and then more gradually. With 400 µM Cu(OP)(2), 90% of the carrier was modified. With a decrease in the amount of AAC, on treatment with Cu(OP)(2) up to 100 µM, the level of (AAC)(2) 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)(2) concentration, attaining a plateau level of 10% of the original amount of AAC at 400 µM Cu(OP)(2). Similar changes were observed in the Coomassie Blue staining intensities of the AAC and (AAC)(2) 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)(2). The intensities of immunostained bands of AAC, (AAC)(2), 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)(2) 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)(2) caused formation of mainly (AAC)(2) 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)(2) 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)(2) concentrations of <100 µM at 0 °C, these mild conditions were suitable for quantitative analyses of the effect of Cu(OP)(2). As shown in Fig. 4, the 60-kDa band of (AAC)(2) formed by treatment of the particles with 100 µM Cu(OP)(2) 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)(2). These results indicate that Cu(OP)(2) 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)(2), no 60-kDa (AAC)(2) 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)(2) 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)(2) 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)(2) and the effect of this cross-linking on ADP transport. For this, we incubated submitochondrial particles with 100 µM Cu(OP)(2) 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)(2). 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)(2) 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)(2) 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)(2) for up to 60 min resulted in no appreciable change in the 30-kDa band (data not shown), showing that Cu(OP)(2), 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)(2) on the ADP/ATP carrier and its ADP transport activity in bovine heart submitochondrial particles. Submitochondrial particles were treated with 100 µM Cu(OP)(2) 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 [^3H]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 [^3H]ADP transported was determined (B). Results are means ± S.D. for three separate runs.



Identification of Cysteine Residues Associated with Disulfide Bridge Formation

To identify the cysteine residues responsible for the formation of the intermolecular disulfide bond in the ADP/ATP carrier catalyzed by Cu(OP)(2), we isolated 60-kDa (AAC)(2) from the particles after treatment with 100 µM Cu(OP)(2) for 20 min at 0 °C, conditions under which formation of the intermolecular cross-linkage was highest (cf.Fig. 5). Then we treated the particles with NEM to alkylate residual free SH groups and solubilized them with Triton X-100. The solubilized preparation was passed through hydroxylapatite gel, and (AAC)(2) in the flow-through fraction was precipitated with acetone, solubilized, and denatured with guanidine HCl containing NEM to alkylate free SH groups completely. The preparation was then subjected to gel filtration with 63% acetonitrile containing 0.05% trifluoroacetic acid to isolate (AAC)(2) (Fig. 6A). As a control, the ADP/ATP carrier without Cu(OP)(2) treatment was also isolated from the particles by the same procedure. (AAC)(2) isolated from particles treated with Cu(OP)(2) and AAC isolated from particles without Cu(OP)(2) treatment each gave a single band on SDS-PAGE (Fig. 6B) and were used for further analyses.


Figure 6: Isolation of the ADP/ATP carrier and its intermolecular cross-linked dimer formed by treatment with Cu(OP)(2) from bovine heart submitochondrial particles. Submitochondrial particles treated with 100 µM Cu(OP)(2) 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 times 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)(2) 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)(2) 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)(2) treatment were used as references. Fractions of AAC and (AAC)(2) 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)(2) are referred to as RC-AAC and RC-(AAC)(2), 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)(2), (AAC)(2) 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)(2) 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)(2) preparations are referred to as RC-AAC and RC-(AAC)(2), 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)(2) 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)(2) 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.

Effects of Transport Inhibitors on Formation of the Disulfide Bridge

CATR and BKA inhibit transport by acting from the cytosolic and matrix sides, respectively. The effects of these inhibitors on cross-linking of the carrier were examined by incubation of submitochondrial particles first with these inhibitors at various concentrations for 10 min at 0 °C and then with 50 µM Cu(OP)(2) for 10 min at 0 °C. As shown in Fig. 8, addition of up to 40 µM BKA to the submitochondrial particles from the matrix side, which completely inhibits transport, did not inhibit the formation of disulfide bridges. In contrast, CATR added from the cytosolic side, but not from the matrix side, completely inhibited the formation of (AAC)(2).


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)(2) for 10 min at 0 °C. Submitochondrial particles (SMP) without treatment with Cu(OP)(2) 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)(2) 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)(2) at various concentrations of BKA and CATR.



Next, the effects of 50 µM Cu(OP)(2) 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)(2) at 0 °C. The time courses of changes in Coomassie Blue staining intensities of AAC and (AAC)(2) are shown in Fig. 9. The decrease in the amount of AAC and the increase in the amount of (AAC)(2) were more gradual than those on treatment with 100 µM Cu(OP)(2) (cf.Fig. 5). As expected, BKA added from the matrix side had no effect on the formation of (AAC)(2), but CATR added from the cytosolic side completely inhibited loss of AAC and formation of (AAC)(2) even on long incubation (60 min) with Cu(OP)(2). 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)(2).


Figure 9: Time courses of formation of the intermolecular cross-linked ADP/ATP carrier on treatment with Cu(OP)(2) 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)(2) 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)(2)) were determined. Experimental conditions were as described for Fig. 8.




DISCUSSION

The cross-linking reagent Cu(OP)(2) 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)(2) may be different from that in the isolated preparation. In this study, we confirmed that Cu(OP)(2) 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)(2), 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)(2) 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)(2) 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)(2). 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)(2) (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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 81-886-33-5195.

(^1)
The abbreviations used are: NEM, N-ethylmaleimide; Cu(OP)(2), copper-o-phenanthroline complex; CATR, carboxyatractyloside; HPLC, high performance liquid chromatography; BKA, bongkrekic acid; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; AAC, ADP/ATP carrier; (AAC)(2), intermolecular cross-linked AAC; AAC`, intramolecular cross-linked AAC; RCAM, AAC reduced and carboxamidomethylated; RNEM, AAC reduced and modified with NEM; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid.


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