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acylcarnitine carrier to membrane-impermeable SH reagents
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Biochimica et Biophysica Acta 1767 (2007) 1331 – 1339 www.elsevier.com/locate/bbabio
Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents Nicola Giangregorio a,b,1 , Annamaria Tonazzi a,b,1 , Cesare Indiveri c , Ferdinando Palmieri a,b,⁎ a
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy b CNR Institute of Biomembranes and Bioenergetics, via Orabona 4, 70125 Bari, Italy c Department of Cellular Biology, University of Calabria, 87036 Arcavacata di Rende, Italy Received 3 July 2007; received in revised form 29 August 2007; accepted 30 August 2007 Available online 18 September 2007
Abstract During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimidetreated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation. © 2007 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Carnitine/acylcarnitine carrier; Site-directed mutagenesis; Membrane transport; Thiol reagent
1. Introduction The mitochondrial carnitine/acylcarnitine carrier (CAC) plays an essential role in fatty acid β-oxidation because it
Abbreviations: CAC, Carnitine/acylcarnitine carrier; MTSES, Sodium(2sulfonatoethyl)-methane-thiosulfonate; MTSEA, Sodium(2-aminoethyl)-methane-thiosulfonate; NEM, N-ethylmaleimide; Pipes, 1,4-piperazinediethanesulfonic acid; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis ⁎ Corresponding author. Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy. Tel.: +39 0 805443374; fax: +39 0 805442770. E-mail address: [email protected] (F. Palmieri). 1 These authors contributed equally to this work. 0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2007.08.010
transports acylcarnitines into the mitochondria in exchange for free carnitine [1]. The CAC, encoded in man by the gene SLC25A20, which maps to chromosome 3p21.31 [2], and in yeast by the gene CRC [3], belongs to a large family of related transport proteins called the mitochondrial carrier family [4–9]. All primary structures of the family members consist of three tandemly repeated homologous domains of about 100 amino acids in length. Each of the three repeats contains two predicted membrane-spanning regions and a characteristic signature motif P-X-[DE]-X-X-[RK]. The significant sequence conservation in the mitochondrial carrier family suggests that the main structural fold is similar for all carriers and that the specific recognition of substrates is coupled to a common structural mechanism of transport [10].
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The function of the CAC has been extensively investigated in mitochondria and in reconstituted liposomes ([11,12] and references therein). This carrier catalyses carnitine/acylcarnitine and carnitine/carnitine antiport efficiently and, in the absence of counter-substrate, 10-fold slower carnitine uniport [13]. The transporter is inserted unidirectionally in liposomes with a rightside out orientation compared to mitochondria [14,15]. A single transport affinity for carnitine was found on each side of the liposomal membrane with the Km value for external carnitine being several times lower than that for internal carnitine [14,15]. Both the homologous carnitine/carnitine exchange and the heterologous carnitine/acylcarnitine exchange occur via a pingpong transport mechanism, implying that only one substratebinding site (not necessarily an identical ensemble of amino acids) is exposed alternatively to the external or the internal side of the carrier and that the concentration of the substrate on one side of the membrane influences the apparent affinity for the substrate on the other side [14]. The structure/function relationship of rat CAC has been investigated by site-directed mutagenesis of the six native Cys residues. None of these cysteines is essential for carrier function [15]. Cys-136 is the major target of SH reagents and is accessible to membraneimpermeable SH reagents from the external (cytosolic) side of the proteoliposomes [15]. Furthermore, it was suggested that Cys-58, Cys-136, and Cys-155 are or come close to each other during certain steps of the catalytic cycle [12]. It is generally accepted that, during their catalytic transport cycle, mitochondrial carriers undergo a transition between the cytoplasmic-state (c-state) and matrix-state (m-state), in which the substrate-binding site faces the cytosolic side and matrix side, respectively [16,17]. In the case of the ADP/ATP carrier, the prototype of the mitochondrial carriers, the structural properties of the c- and m-states have been investigated by sitedirected mutagenesis and/or chemical modification using two powerful and state-specific inhibitors: carboxyatractyloside, that locks the carrier in the c-state, and bongkrekic acid, that locks the carrier in the m-state [18–22]. The only available highresolution structure is that of the ADP/ATP carrier in complex with carboxyatractyloside, which corresponds most likely to the c-state [23]. This structure consists of a barrel of six transmembrane α-helices (H1–H6) and three short α-helices (h12, h34 and h56) situated in the matrix and parallel to the membrane plane. The prolines of the signature motif sharply kink the odd-numbered transmembrane α-helices, and the charged residues of the motif form a salt bridge network that closes the cavity where carboxyatractyloside is located on the matrix side of the protein [23]. This structure has considerably improved our understanding of the mitochondrial carrier structure/function relationships. However, further studies are necessary to elucidate the structural changes occurring during the transition between the c- and m-states and to better understand the mechanism of substrate translocation through the mitochondrial carriers. In this work, we probed the accessibility/chemical reactivity of the rat CAC native cysteine residues, Cys-58, Cys-136 and Cys-155, to membrane-impermeable SH reagents under variation of external or internal substrate concentrations, i.e., under
conditions that change the ratio between the c- and m-states of the carrier. A strong influence of the conformational state of the carrier on the accessibility of Cys-136 and Cys-155 to these reagents is observed. 2. Materials and methods 2.1. Materials l-[methyl-3H]carnitine and Sephadex G-25 (PD-10) were purchased from Amersham, MTS reagents from Toronto Research (North York, Ontario, Canada), Sephadex G-50, G-75 and G-200, egg-yolk phospholipids (l-αphosphatidylcholine from fresh turkey egg yolk), Pipes, Triton X-100, cardiolipin, l-carnitine and N-dodecanoylsarcosine (sarcosyl) from Sigma. All other reagents were of analytical grade.
2.2. Site-directed mutagenesis, overexpression and isolation of the CAC proteins The coding region for the rat CAC was amplified from total rat liver cDNA as described previously [1]. The Cys/Ser replacements were constructed with complementary mutagenic primers using the overlap extension method [24] and the High Fidelity PCR System (Roche). The PCR products were purified using the Gene Clean Kit (La Jolla), digested with NdeI and HindIII (restriction sites added at the 5′ end of forward and reverse primers, respectively) and ligated into the pMW7 expression vector. All mutations were verified by DNA sequencing and, except for the desired base changes, all of the sequences were identical to that of rat CAC cDNA. The resulting plasmids were transformed into Escherichia coli C0214 [15]. Bacterial overexpression, isolation of the inclusion body fraction, and solubilization and purification of the wild-type CAC and mutant CAC proteins were performed as described previously [15].
2.3. Reconstitution of CAC and CAC mutants in liposomes The recombinant proteins were reconstituted into liposomes as described previously [25]; however, the concentration of intraliposomal carnitine was varied as indicated in the legends to figures. The external substrate was removed from proteoliposomes on Sephadex G-75 columns.
2.4. Transport measurements Transport at 25 °C was started by adding [3H]carnitine, at the concentrations indicated in the legends to figures, to proteoliposomes and terminated by the addition of 1.5 mM NEM [15,25]. In the case of the mutant C23S/C89S/C136S/ C155S/C283S containing only Cys-58, the reaction was rapidly quenched by passing the samples through Sephadex G-50 at 2 °C. In controls, the inhibitor was added together with the labeled substrate, according to the inhibitor stop method [25]. Finally, the external substrate was removed by chromatography on Sephadex G-50 columns, and the radioactivity in the liposomes was measured. The initial transport rate was calculated from the radioactivity taken up by proteoliposomes after 1 min (in the initial linear range of substrate uptake). The experimental values were corrected by subtracting control values. All of the transport activities were determined by taking into account the efficiency of reconstitution (i.e., the share of successfully incorporated protein).
2.5. Other methods SDS-PAGE was performed according to Laemmli, as described previously [15]. The amount of recombinant protein was estimated on Coomassie Bluestained SDS-PAGE gels by the Bio-Rad GS-700 Imaging Densitometer equipped with the Bio-Rad Multi-Analist software using bovine serum albumin as standard. The extent of incorporation of the recombinant protein into liposomes was determined as described in Phelps et al. [26] with some modifications [15]. The homology model of the rat CAC was built based upon the structure of the bovine AAC1 [23] by using the computer application Swiss PDB Viewer [27]. A critical determinant in the accuracy of the comparative
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modeling is the quality of the pairwise alignment between the CAC and AAC1 template sequences. AAC1 and CAC were added to sequences from the mitochondrial carrier family and a multiple sequence alignment was generated by CLUSTALW. The secondary structure of AAC1 was used to weight the gap penalties as described previously [10]. One thousand steps of energy minimization were performed to generate 50 minimized CAC structures. The structural properties of the CAC models with the best energy function were evaluated using protein analysis tools available on the WHAT IF Web server [28]. Final models were examined with PyMOL, VMD and Swiss PDB Viewer and, where side-chain packing led to clashes, alternative side-chain rotamers were evaluated.
3. Results 3.1. Transport mechanism of CAC mutants The CAC contains six cysteines: Cys-23, Cys-58, Cys-89, Cys-136, Cys-155 and Cys-283. Wild-type CAC and CAC mutants, containing the combinations Cys-58/Cys-136, Cys-58/ Cys-155 and Cys-136/Cys-155 and the other four native cysteines replaced by serine, were overexpressed in E. coli, purified and reconstituted into liposomes to measure the transport rate of carnitine. In the comparative model of CAC,
Fig. 2. Bi-substrate analysis of the carnitine/carnitine antiport reaction catalyzed by reconstituted CAC mutants. Lineweaver–Burk plots showing the dependence of the carnitine/carnitine antiport rate on external carnitine concentration at three different internal carnitine concentrations. Transport was started by adding the indicated [3H]carnitine concentrations to liposomes reconstituted with the C58 + C136 mutant (A) or the C58 + C155 mutant (B) and preloaded internally with 2.5 mM (□), 7 mM (•) and 15 mM (○) unlabeled carnitine. Incubation time was 1 min. The Cys residues present in the mutants are shown. Similar results were obtained in three independent experiments in duplicate.
Fig. 1. Structural model of the CAC. Ribbon diagrams viewing the carrier from the lateral side. The transmembrane α-helices are colored as follows: H1, red; H2, tan (light brown); H3, green; H4, emerald-green; H5, cyan; and H6, blue. Purple surfaces highlight the SH groups of the Cys residues indicated by their number in the CAC sequence. Yellow surfaces highlight the salt bridge network between residues D32-K135, K35-D231 and E132-K234.
which was built using the structure of the ADP/ATP carrier in c-state as template [23], Cys-58, Cys-136 and Cys-155 are localised below the salt bridge network that closes the central pore of the α-helical bundle on the matrix side preventing translocation of the substrate (Fig. 1). The reconstituted CAC purified from rat liver mitochondria functions according to a ping-pong mechanism [14]. In this mechanism [29–32], only one substrate-binding site exists in the carrier, which is alternatively exposed to the external side (outward-facing conformation) or to the internal site (inwardfacing conformation) of the membrane. To ascertain whether the recombinant cysteine mutant carriers follow the same kinetic mechanism, the carnitine/carnitine exchange activity mediated by these proteins was measured by varying both external and internal substrate concentrations. When the kinetic data were analyzed in Lineweaver–Burk plots (Fig. 2A and B), showing the dependence of the transport rate on external carnitine at
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three different internal concentrations (2.5, 7.0 and 15.0 mM), a parallel pattern of straight lines was obtained with the CAC four Cys-replacement mutants C23S/C89S/C155S/C283S (containing Cys-58 and Cys-136) and C23S/C89S/C136S/C283S (containing Cys-58 and Cys-155). The external Km for carnitine of mutant C23S/C89S/C155S/C283S was increased from 0.4 to 2.4 mM (Fig. 2A) and that of the mutant C23S/C89S/C136S/ C283S from 0.1 to 1.0 mM (Fig. 2B) on increasing the internal substrate concentration from 2.5 to 15.0 mM. Clearly, variation of the substrate concentration in the internal compartment strongly influences the apparent transport affinity for the counter-substrate in the external compartment as a consequence of the change in the instant ratio between outward- and inwardfacing conformations of the carrier protein. Furthermore, secondary plots of the slopes and ordinate intercepts of Fig. 2A and B versus the reciprocal concentration of the substrate on the opposite side (not shown) demonstrated that the ratio of Km/Vmax did not change upon variation of the countersubstrate concentration. Similar results were obtained with the wild-type recombinant CAC and with the CAC C23S/C58S/ C89S/C283S mutant containing Cys-136 and Cys-155. Taken together these results show that all these proteins function according to a ping-pong mechanism. 3.2. Accessibility of Cys-58 to SH reagents To investigate the influence of the conformational state of the carrier on the accessibility of Cys-58, Cys-136 or Cys-155 to sulphydryl reagents, it would be ideal to use the five Cysreplacement mutants containing only Cys-58, Cys-136 or Cys155. However, all the CAC five Cys-replacement mutants had been previously found to be inactive [15]. Therefore, we first attempted to improve the activity of these mutants by adding increasing amounts of cardiolipin to the mixture used for their reconstitution into liposomes [11,33,34]. Fig. 3 shows that, although the wild-type CAC protein exhibited maximal activity at 200 μg/ml cardiolipin, the five replacement mutants C23S/ C89S/C136S/C155S/C283S, C23S/C89S/C58S/C155S/C283S and C23S/C58S/C89S/C136S/C283S (containing only Cys-58, Cys-136 and Cys-155, respectively) were virtually inactive at this cardiolipin concentration. At 500 μg/ml cardiolipin, however, the C23S/C89S/C136S/C155S/C283S mutant containing only Cys-58 displayed 40% transport activity compared to the wild-type CAC, whereas the other two 5 replacement mutants still exhibited negligible activity. Consequently, after its reconstitution in the presence of 500 μg/ml cardiolipin, the C23S/C89S/C136S/C155S/C283S mutant was tested for sensitivity to SH reagents. It was found that the carnitine transport activity of the mutant, measured as exchange between 0.1 mM external [3H]carnitine and 2.5 or 15.0 mM internal carnitine, was not affected by 0.5 mM MTSES or MTSEA, whether added outside the proteoliposomes even 10 min before adding the labeled substrate or present inside the proteoliposomes, i.e., during the reconstitution procedure (data not shown). Similarly, no effect of 0.5 mM MTSES or MTSEA on the C23S/C89S/ C136S/C155S/C283S mutant was observed when the activity was measured by adding 0.2 or 5 mM [3H]carnitine to pro-
Fig. 3. Dependence of CAC mutant activity on cardiolipin. Liposomes reconstituted with wild-type CAC (○), the C58 (•), C136 (▽) or C155 (▴) CAC mutant, in the presence of the indicated concentrations of cardiolipin, were preloaded internally with 13 mM carnitine. Transport was started with 0.1 mM [3H]carnitine and terminated after 10 min. Similar results were obtained in three independent experiments in duplicate.
teoliposomes containing 5 mM carnitine. In contrast, NEM inhibited the activity of the C23S/C89S/C136S/C155S/C283S mutant by about 80% at 0.5 mM when present in the internal compartment and had no effect up to 2.0 mM when preincubated externally for 10 min with the proteoliposomes (data not shown). These results indicate that Cys-58 is not accessible to MTSES and MTSEA, whereas it is accessible to NEM but only from the internal side of the reconstituted protein. 3.3. Accessibility of Cys-136 and Cys-155 to MTS reagents under variation of internal and external substrate concentrations Given that the five cysteine-replacement mutants C23S/ C89S/C58S/C155S/C283S and C23S/C58S/C89S/C136S/ C283S are inactive and Cys-58 is insensitive to externally added MTSES and MTSEA, the accessibility of Cys-136 and Cys-155 to these reagents was investigated in proteoliposomes reconstituted with one of the four Cys-replacement mutants, C23S/C89S/C155S/C283S, C23S/C89S/C136S/C283S and C23S/C58S/C89S/C283S, which contain the combinations Cys-58/Cys-136, Cys-58/Cys-155 and Cys-136/Cys-155, respectively. In a first set of experiments, MTSES or MTSEA was added together with 0.1 mM [3H]carnitine to proteoliposomes pre-loaded internally with two widely different carnitine concentrations (2.5 and 15.0 mM) and the initial carnitine/ carnitine exchange rate was measured. The residual transport activity in the presence of MTSES is shown in Fig. 4A–C. With all three mutants the inhibition of the transport rate by MTSES was clearly greater with 15.0 mM than with 2.5 mM internal carnitine. Furthermore, the mutant containing the Cys-58/Cys155 combination was much less sensitive to MTSES than the mutant containing the Cys-58/Cys-136 combination. Thus, half maximal inhibition of C23S/C89S/C136S/C283S was obtained
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mutants more in the presence of 15 mM than 2.5 mM internal carnitine and also inhibited more Cys-58/Cys-136 than Cys-58/ Cys-155 (data not shown). With the Cys-58/Cys-136 mutant, 4 μM MTSEA yielded total inhibition in the presence of 15 mM
Fig. 4. Effect of MTSES on the activity of C58 + C136, C58 + C155 and C136 + C155 mutants at different intraliposomal carnitine concentrations. Liposomes reconstituted with the C58 + C136 (A), C58 + C155 (B) or C136 + C155 (C) mutant were preloaded internally with 2.5 mM (○) or 15 mM (•) carnitine. Transport was started by adding 0.1 mM [3H]carnitine together with the indicated concentrations of MTSES and stopped after 1 min. Results are expressed as percent of residual activity (i.e., percent of activity in the presence of MTSES with respect to the control value without inhibitor). The data represent means ± SD of at least three independent experiments in duplicate. The Cys residues present in the CAC mutants are shown.
at a concentration more than one order of magnitude higher than that required for half maximal inhibition of C23S/C89S/C155S/ C283S. Similarly to MTSES, MTSEA inhibited the initial carnitine/carnitine exchange rate catalyzed by the same three
Fig. 5. Effect of MTSES on the activity of the C58 + C136, C58 + C155 and C136 + C155 mutants at different external carnitine concentrations. Liposomes were reconstituted with the C58 + C136 (A), C58 + C155 (B) or C136 + C155 (C) mutant and preloaded internally with 5 mM carnitine. Transport was started by adding 0.2 mM (□) or 5 mM (n) [3H]carnitine together with the indicated concentrations of MTSES. Incubation time was 1 min. Results are expressed as percent of residual activity (i.e., percent of activity in the presence of MTSES with respect to the control value without inhibitor). The data represent means ± SD of at least three independent experiments in duplicate. The Cys residues present in the CAC mutants are shown.
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internal carnitine and 8 μM MTSEA 93% inhibition in the presence of 2.5 mM internal carnitine; with the Cys-58/Cys-155 mutant, 8 μM MTSEA yielded 67% inhibition in the presence of 15 mM internal carnitine and 47% inhibition in the presence of 2.5 mM. All these results indicate that Cys-136 is more accessible than Cys-155 to MTSES and MTSEA and that the accessibility of both Cys residues is greater at the higher internal carnitine concentration, which favours the inward conformation of the protein. In other experiments, the effect of MTSES on the activity of the four Cys-replacement mutants C23S/C89S/C155S/C283S, C23S/C89S/C136S/C283S and C23S/C58S/C89S/C283S was measured in the presence of two different external carnitine concentrations (0.2 and 5.0 mM) at a constant internal concentration of 5.0 mM. MTSES inhibited all three mutants more with 0.2 mM than with 5.0 mM external carnitine (Fig. 5A–C). Results similar to those reported in Fig. 5 were obtained using MTSEA at concentrations up to 8 μM instead of MTSES (data not shown). Therefore, the accessibility of both Cys-136 and Cys-155 decreases upon increasing the external concentration, which favours the outward conformation of the carrier. Furthermore, as observed in the experiments where the substrate concentration was varied inside the proteoliposomes, the mutant containing the Cys-58/Cys-155 combination was less sensitive to MTSEA, and especially to MTSES, than the mutant containing the Cys-58/Cys-136 combination. It should be noted that the concentrations of substrate used outside and inside the
proteoliposomes in the experiments of Figs. 4 and 5 were chosen on the basis of the affinity of the carrier for carnitine on each side of the liposomal membrane [14,15] and on the fact that low internal and high external concentrations cannot be used simultaneously for reliable measurements of radioactive substrate uptake [25]. 3.4. Effect of intraliposomal carnitine concentration on Cys labeling by fluorescein-5-maleimide To further support the dependence of Cys-136 and Cys-155 accessibility on the intraliposomal substrate concentration, the effect of fluorescein-5-maleimide, another impermeable SH reagent [35], was investigated. In these experiments the mutants C23S/C58S/C89S/C283S, C23S/C89S/C155S/C283S and C23S/C89S/C136S/C283S containing Cys-136/Cys-155, Cys58/Cys-136 and Cys-58/Cys-155, respectively, were treated with fluorescein-5-maleimide in reconstituted liposomes under the two conditions of internal substrate concentration. Afterward, the mutant proteins were extracted, separated by SDSPAGE and either analyzed for fluorescence (Fig. 6A) or stained with silver nitrate (Fig. 6B). All three CAC mutants were more fluorescent when extracted from the proteoliposomes containing 15 mM internal carnitine than from the proteoliposomes containing 2.5 mM carnitine, indicating that the amount of bound fluorescein-5-maleimide depends on the internal carnitine concentration. In parallel experiments, reconstituted liposomes
Fig. 6. Influence of intraliposomal carnitine concentration on the labeling and inhibition of CAC mutants by fluorescein-5-maleimide. Liposomes reconstituted with the C136 + C155, C58 + C136 or C58 + C155 mutant and preloaded internally with 2.5 mM or 15 mM unlabeled carnitine were incubated with fluorescein-5maleimide, at 10 μM (A and B) and at the indicated concentrations (C) for 1 min at 25 °C; the reaction was then quenched by adding 10 mM DTE. The proteoliposomes were passed through Sephadex G-75 columns and ultracentrifuged at 110,000×g for 45 min at 4 °C; the pellets were suspended in 2% SDS, precipitated with acetone and subjected to SDS-PAGE. (A) Fluorescence of the UV-illuminated SDS-PAGE gel; (B) silver staining of the same gel; (C) inhibition of the carnitine/carnitine exchange activity by fluorescein-5-maleimide. Transport was started by adding 0.1 mM [3H]carnitine together with fluorescein-5-maleimide. Incubation time was 1 min. Results are expressed as percent of residual activity (i.e., percent of activity in the presence of fluorescein-5-maleimide with respect to the control value without inhibitor). The data represent means ± SD of three independent experiments in duplicate. The Cys residues present in the CAC mutants are shown. Abbreviation: FM, fluorescein-5-maleimide.
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treated with fluorescein-5-maleimide under the same conditions as in Fig. 6A were tested for residual transport activity (Fig. 6C). With all the mutants transport inhibition by fluorescein-5maleimide was increased by the presence of the higher intraliposomal concentration of carnitine, in agreement with the labeling results. Furthermore, virtually no labeling or inhibition by fluorescein-5-maleimide up to 10 μg/ml was detected in the case of the mutant C23S/C89S/C136S/C155S/C283S containing only Cys-58 reconstituted in the presence of 500 μg/ml cardiolipin (data not shown). 4. Discussion The CAC functions according to a ping-pong mechanism that implies the existence of two opposite conformations in which the substrate-binding site is alternatively exposed towards the cytosol (c-state or outward-facing conformation) or towards the matrix (m-state or inward-facing conformation). Unfortunately for CAC there are no side-specific ligands available that fix CAC in one of these conformations, as has been done with the ADP/ATP carrier by the addition of carboxyatractyloside or bongkrekic acid [17,36]. Here we took advantage of the transport mechanism of CAC, according to which the ratio between the inward- and the outward-facing conformation of the carrier can be varied by changing the concentration of the substrate inside or outside the proteoliposomes, in the presence of a fixed substrate concentration on the opposite side. Furthermore, the reconstituted CAC proteins were incubated with SH reagents only for the time interval used to measure the initial transport rate in order to limit the number of the catalytic cycles as much as possible. The experimental data reported above show that Cys-136 and Cys-155 of the reconstituted CAC become more accessible to the membrane-impermeable MTSES, MTSEA or fluorescein-5maleimide when the intraliposomal carnitine concentration is increased, therefore favouring the m-state of the carrier. Alternatively, these cysteines become less accessible when the concentration of carnitine is increased in the external compartment of the proteoliposomes, i. e., when the carrier is in the c-state. The three cysteines whose accessibility has been investigated here, Cys-58, Cys-136 and Cys-155, are located below the salt bridge network that in the c-state of the carrier closes the substrate translocation pathway on the matrix side (Fig. 1). Given that Cys-136 is located immediately below the salt bridge between K135 and D32 of the ionic network (Fig. 1), the greater accessibility of Cys-136 to externally added impermeable reagents when the majority of CAC molecules are in the mstate is most likely caused by the opening of the salt bridge network that must occur during the transition from the c- to the m-state. The conformational changes involved in substrate translocation are not yet known. However, it is thought that in the catalytic cycle of the ADP/ATP carrier the inter-repeat salt bridges between the charged residues of the three signature motifs break causing their rearrangement into intra-repeat salt bridges [37–39] and the stretching of the odd-numbered helices (H1, H3 and H5) [23] leading to the opening of the translocation
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channel. The corresponding inter-repeat salt bridges in rat CAC are D32-K135, E132-K234 and D231-K35, and the intra-repeat salt bridges are D32-K35, E132-K135 and D231-K234. The hypothesized straightening of the odd-numbered helices, in particular of H3, would make Cys-155 more accessible to externally added MTSES, MTSEA and fluorescein-5-maleimide in the m-state, although Cys-155 may be still partially screened by H3 in the m-state. This possibility may account for the lower sensitivity of Cys-155 toward methanethiosulfonate reagents compared to that of Cys-136. Furthermore, given that MTSES is negatively charged, the positive charge density of R133 and K135, that belong to H3 and are close to Cys-155, may diminish the interaction between MTSES and Cys-155. In a previous paper, it was shown that the reaction of externally added MTSES with Cys-136 of reconstituted CAC is prevented by the addition of carnitine to proteoliposomes [15]. It was suggested that Cys-136 is localised at or near the substratebinding site. Therefore, the substrate would impede the interaction of MTSES with Cys-136 either by direct competition with Cys-136 or by steric hindrance due to binding with side chains close to Cys-136. The results reported in this work, and in particular those obtained in the presence of different intraliposomal carnitine concentrations, favour the alternative explanation, i.e., that externally added substrate induces a conformational change of the carrier protein, thereby decreasing the binding of the inhibitors MTSES, MTSEA and fluorescein5-maleimide to a site different from that of the substrate. In this context it is worth mentioning that acetylcarnitine has been proposed to bind to F90, R180, D181 and R280 of the S. cerevisiae CAC [40], corresponding to the residues F86, R178, D179 and R275 of the rat CAC that are found in the cavity at the midpoint of the membrane. Unfortunately, it is not possible to ascertain whether Cys-136 is completely inaccessible in the cstate because the experimental approach used in this work does not allow to fix all of the carrier molecules in the same conformation. Furthermore, it should be stressed that the conformational change in the carrier can affect not only the accessibility but also the reactivity of Cys-136 and Cys-155. This may occur in different ways; for example, a change in the pK of the side chain of cysteine may take place when the microenvironment of the sulfhydryl group becomes more hydrophobic or hydrophilic. The results obtained using the CAC mutant containing only Cys-58 clearly show that Cys-58 behaves differently from Cys136 and Cys-155 because it is not accessible to MTSES, MTSEA and fluorescein-5-maleimide in all the experimental conditions used. This observation can be explained by the CAC homology model (Fig. 1), as Cys-58 is sterically hindered to externally added hydrophilic reagents being efficiently screened by H1. Unlike Cys-155, which is located at the beginning of h34, Cys-58 is located approximately at the midpoint of h12, and therefore the stretching of H1 occurring during the transition from the c-state to the m-state does not increase accessibility to Cys-58 as does the stretching of H3 to Cys-155. It could be argued that MTSES, MTSEA and fluorescein-5-maleimide bind Cys-58 without inhibiting the carrier. An argument in favour of the conclusion that they do not react with Cys-58 is the
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observation that when present inside the proteoliposomes, NEM inhibits the CAC mutant containing Cys-58 unlike Cys-less CAC. The accessibility of Cys-58 to NEM with respect to the hydrophilic reagents is favoured by the fact that this residue is surrounded by Y52, F59 and F64 that form an aromatic pocket. The results of the present study show that the matrix α-helix of the CAC that connects transmembrane helices H3 and H4, and contains Cys-136 and Cys-155, undergoes conformational changes during the catalytic cycle of the carrier. Our data are in line with the previous finding [21] that the corresponding region of the ADP/ATP carrier is accessible to externally added trypsin in a conformational-sensitive manner, being cleaved at the Lys178–Thr179 bond in the carrier-bongkrekic acid complex but not in the carrier-carboxyatractyloside complex. Indeed, positions 178 and 179 of the ADP/ATP carrier correspond to residues 157 and 158 of the CAC that are very close to Cys-155. This suggests rather similar conformational changes in the central matrix loop of the two mitochondrial carrier proteins. Acknowledgements This work was supported by grants from the Ministero dell'Università e della Ricerca (MUR), the Center of Excellence in Genomics (CEGBA), and EC contract LSHM-CT-2004503116. References [1] C. Indiveri, V. Iacobazzi, N. Giangregorio, F. Palmieri, The mitochondrial carnitine carrier protein: cDNA cloning, primary structure, and comparison with other mitochondrial transport proteins, Biochem. J. 321 (1997) 713–719. [2] V. Iacobazzi, M.A. Naglieri, C.A. Stanley, J.A.R. Wanders, F. Palmieri, The structure and organization of the human carnitine/acylcarnitine translocase (CACT) gene, Biochem. Biophys. Res. Commun. 252 (1998) 770–774. [3] L. Palmieri, F.M. Lasorsa, V. Iacobazzi, M.J. Runswick, F. Palmieri, J.E. Walker, Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae, FEBS Lett. 462 (1999) 472–476. [4] F. Palmieri, The mitochondrial transporter family (SLC25): physiological and pathological implications, in: M.A. Hediger (Ed.), The ABC of Solute Carriers, Pflugers Arch.-Eur. J. Physiol., vol. 447, 2004, pp. 689–709. [5] R. Krämer, F. Palmieri, in: L. Ernster (Ed.), Molecular Mechanisms in Bioenergetics, Elsevier Science Publishers, B.V., Amsterdam, 1992, pp. 359–384. [6] J.E. Walker, The mitochondrial transporter family, Curr. Opin. Struck. Biol. 2 (1992) 519–526. [7] E.R. Kunji, The role and structure of mitochondrial carriers, FEBS Lett. 564 (2004) 239–244. [8] N. Picault, M. Hodges, L. Palmieri, F. Palmieri, The growing family of mitochondrial carriers in Arabidopsis, Trends Plant Sci. 9 (2004) 138–146. [9] F. Palmieri, G. Agrimi, E. Blanco, A. Castegna, M.A. Di Noia, V. Iacobazzi, F.M. Lasorsa, C.M. Marobbio, L. Palmieri, P. Scarcia, S. Todisco, A. Vozza, J. Walker, Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins, Biochim. Biophys. Acta 1757 (2006) 1249–1262. [10] A.R. Cappello, R. Curcio, D.V. Miniero, I. Stipani, A.J. Robinson, E.R.S. Kunji, F. Palmieri, Functional and structural role of amino acid residues in the even-numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate carrier, J. Mol. Biol. 363 (2006) 51–62.
[11] C. Indiveri, A. Tonazzi, F. Palmieri, Identification and purification of the carnitine carrier from rat liver mitochondria, Biochim. Biophys. Acta 1020 (1990) 81–86. [12] A. Tonazzi, N. Giangregorio, C. Indiveri, F. Palmieri, Identification by site-directed mutagenesis and chemical modification of three vicinal cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter, J. Biol. Chem. 280 (2005) 19607–19612. [13] C. Indiveri, A. Tonazzi, F. Palmieri, Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria, Biochim. Biophys. Acta 1069 (1991) 110–116. [14] C. Indiveri, A. Tonazzi, F. Palmieri, The reconstituted carnitine carrier from rat liver mitochondria: evidence for a transport mechanism different from that of the other mitochondrial translocators, Biochim. Biophys. Acta 1189 (1994) 65–73. [15] C. Indiveri, N. Giangregorio, V. Iacobazzi, F. Palmieri, Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136, Biochemistry 41 (2002) 8649–8656. [16] M. Klingenberg, Molecular aspects of the adenine nucleotide carrier from mitochondria, Arch. Biochem. Biophys. 270 (1989) 1–14. [17] C. Fiore, V. Trezeguet, A. Le Saux, P. Roux, C. Schwimmer, A.C. Dianoux, F. Noel, G.J.-M. Lauquin, G. Brandolin, P.V. Vignais, The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects, Biochimie 80 (1998) 137–150. [18] E. Majima, K. Ikawa, M. Takeda, M. Hashimoto, Y. Shinohara, H. Terada, 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, J. Biol. Chem. 270 (1995) 29548–29554. [19] M. Hashimoto, E. Majima, S. Goto, Y. Shinohara, H. Terada, Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides, Biochemistry 38 (1999) 1050–1056. [20] Y. Kihira, A. Iwahashi, E. Majima, H. Terada, Y. Shinohara, Twisting of the second transmembrane α-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states, Biochemistry 43 (2004) 15204–15209. [21] C. Dahout-Gonzalez, C. Ramus, E.P. Dassa, A.-C. Dianoux, G. Brandolin, Conformation-dependent swinging of the matrix loop m2 of the mitochondrial Saccharomyces cerevisiae ADP/ATP carrier, Biochemistry 44 (2005) 16310–16320. [22] A. Iwahashi, Y. Kihira, E. Majima, H. Terada, N. Yamazaki, M. Kataoka, Y. Shinohara, The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state, Mitochondrion 6 (2006) 245–251. [23] E. Pebay-Peyroula, C. Dahout-Gonzalez, R. Kahn, V. Trezeguet, G.J. Lauquin, G. Brandolin, Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside, Nature 426 (2003) 39–44. [24] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pullen, L.R. Pease, Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77 (1989) 51–59. [25] F. Palmieri, C. Indiveri, F. Bisaccia, V. Iacobazzi, Methods Enzymol. 260 (1995) 349–369. [26] A. Phelps, C. Briggs, L. Mincone, H. Wohlrab, Mitochondrial phosphate transport protein. Replacements of glutamic, aspartic, and histidine residues affect transport and protein conformation and point to a coupled proton transport path, Biochemistry 35 (1996) 10757–10762. [27] N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling, Electrophoresis 18 (1997) 2714–2723. [28] G. Vriend, WHAT IF: a molecular modeling and drug design program, J. Mol. Graph. 8 (1990) 52–56. [29] W.W. Cleland, in: P.D. Boyer (Ed.), The Enzymes, vol. 2, Academic Press, New York, 1970, pp. 1–65. [30] I.H. Segel, Enzyme kinetics, Wiley, New York, 1975, pp. 274–320. [31] W. Furuya, T. Tarshis, F.-Y. Law, P.A. Knauf, J. Gen. Physiol. 83 (1984) 657–681.
N. Giangregorio et al. / Biochimica et Biophysica Acta 1767 (2007) 1331–1339 [32] F. Palmieri, M. Klingenberg, Mitochondrial metabolite transporter family, in: W.J. Lennarz, M.D. Lane (Eds.), Encyclopedia of Biological Chemistry, vol. 2, Elsevier, Oxford, 2004, pp. 725–732. [33] G. Fiermonte, V. Dolce, F. Palmieri, Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria, J. Biol. Chem. 273 (1998) 22782–22787. [34] R.S. Kaplan, J.A. Mayor, D. Brauer, R. Kotaria, D.D. Walters, A.M. Dean, The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway, J. Biol. Chem. 275 (2000) 12009–12016. [35] P.C. Jones, A. Sivaprasadarao, D. Wray, J.B. Findlay, A method for determining transmembrane protein structure, Mol. Membr. Biol. 13 (1996) 53–60.
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[36] M. Klingenberg, Dialectics in carrier research: the ADP/ATP carrier and the uncoupling protein, J. Bioenerg. Biomembranes 25 (1993) 447–457. [37] D.R. Nelson, C.M. Felix, J.M. Swanson, Highly conserved charge-pair networks in the mitochondrial carrier family, J. Mol. Biol. 277 (1998) 285–308. [38] E.R. Kunji, A.J. Robinson, The conserved substrate binding site of mitochondrial carriers, Biochim. Biophys. Acta 1757 (2006) 1237–1248. [39] M. Falconi, G. Chillemi, D. Di Marino, I. D'Annessa, B. Morozzo della Rocca, L. Palmieri, A. Desideri, Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies, Proteins 65 (2006) 681–691. [40] A.J. Robinson, E.R. Kunji, Mitochondrial carriers in the cytoplasmic state have a common substrate binding site, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 2617–2622.
Biochimica et Biophysica Acta 1767 (2007) 1331 – 1339 www.elsevier.com/locate/bbabio
Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents Nicola Giangregorio a,b,1 , Annamaria Tonazzi a,b,1 , Cesare Indiveri c , Ferdinando Palmieri a,b,⁎ a
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy b CNR Institute of Biomembranes and Bioenergetics, via Orabona 4, 70125 Bari, Italy c Department of Cellular Biology, University of Calabria, 87036 Arcavacata di Rende, Italy Received 3 July 2007; received in revised form 29 August 2007; accepted 30 August 2007 Available online 18 September 2007
Abstract During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimidetreated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation. © 2007 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Carnitine/acylcarnitine carrier; Site-directed mutagenesis; Membrane transport; Thiol reagent
1. Introduction The mitochondrial carnitine/acylcarnitine carrier (CAC) plays an essential role in fatty acid β-oxidation because it
Abbreviations: CAC, Carnitine/acylcarnitine carrier; MTSES, Sodium(2sulfonatoethyl)-methane-thiosulfonate; MTSEA, Sodium(2-aminoethyl)-methane-thiosulfonate; NEM, N-ethylmaleimide; Pipes, 1,4-piperazinediethanesulfonic acid; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis ⁎ Corresponding author. Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy. Tel.: +39 0 805443374; fax: +39 0 805442770. E-mail address: [email protected] (F. Palmieri). 1 These authors contributed equally to this work. 0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2007.08.010
transports acylcarnitines into the mitochondria in exchange for free carnitine [1]. The CAC, encoded in man by the gene SLC25A20, which maps to chromosome 3p21.31 [2], and in yeast by the gene CRC [3], belongs to a large family of related transport proteins called the mitochondrial carrier family [4–9]. All primary structures of the family members consist of three tandemly repeated homologous domains of about 100 amino acids in length. Each of the three repeats contains two predicted membrane-spanning regions and a characteristic signature motif P-X-[DE]-X-X-[RK]. The significant sequence conservation in the mitochondrial carrier family suggests that the main structural fold is similar for all carriers and that the specific recognition of substrates is coupled to a common structural mechanism of transport [10].
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The function of the CAC has been extensively investigated in mitochondria and in reconstituted liposomes ([11,12] and references therein). This carrier catalyses carnitine/acylcarnitine and carnitine/carnitine antiport efficiently and, in the absence of counter-substrate, 10-fold slower carnitine uniport [13]. The transporter is inserted unidirectionally in liposomes with a rightside out orientation compared to mitochondria [14,15]. A single transport affinity for carnitine was found on each side of the liposomal membrane with the Km value for external carnitine being several times lower than that for internal carnitine [14,15]. Both the homologous carnitine/carnitine exchange and the heterologous carnitine/acylcarnitine exchange occur via a pingpong transport mechanism, implying that only one substratebinding site (not necessarily an identical ensemble of amino acids) is exposed alternatively to the external or the internal side of the carrier and that the concentration of the substrate on one side of the membrane influences the apparent affinity for the substrate on the other side [14]. The structure/function relationship of rat CAC has been investigated by site-directed mutagenesis of the six native Cys residues. None of these cysteines is essential for carrier function [15]. Cys-136 is the major target of SH reagents and is accessible to membraneimpermeable SH reagents from the external (cytosolic) side of the proteoliposomes [15]. Furthermore, it was suggested that Cys-58, Cys-136, and Cys-155 are or come close to each other during certain steps of the catalytic cycle [12]. It is generally accepted that, during their catalytic transport cycle, mitochondrial carriers undergo a transition between the cytoplasmic-state (c-state) and matrix-state (m-state), in which the substrate-binding site faces the cytosolic side and matrix side, respectively [16,17]. In the case of the ADP/ATP carrier, the prototype of the mitochondrial carriers, the structural properties of the c- and m-states have been investigated by sitedirected mutagenesis and/or chemical modification using two powerful and state-specific inhibitors: carboxyatractyloside, that locks the carrier in the c-state, and bongkrekic acid, that locks the carrier in the m-state [18–22]. The only available highresolution structure is that of the ADP/ATP carrier in complex with carboxyatractyloside, which corresponds most likely to the c-state [23]. This structure consists of a barrel of six transmembrane α-helices (H1–H6) and three short α-helices (h12, h34 and h56) situated in the matrix and parallel to the membrane plane. The prolines of the signature motif sharply kink the odd-numbered transmembrane α-helices, and the charged residues of the motif form a salt bridge network that closes the cavity where carboxyatractyloside is located on the matrix side of the protein [23]. This structure has considerably improved our understanding of the mitochondrial carrier structure/function relationships. However, further studies are necessary to elucidate the structural changes occurring during the transition between the c- and m-states and to better understand the mechanism of substrate translocation through the mitochondrial carriers. In this work, we probed the accessibility/chemical reactivity of the rat CAC native cysteine residues, Cys-58, Cys-136 and Cys-155, to membrane-impermeable SH reagents under variation of external or internal substrate concentrations, i.e., under
conditions that change the ratio between the c- and m-states of the carrier. A strong influence of the conformational state of the carrier on the accessibility of Cys-136 and Cys-155 to these reagents is observed. 2. Materials and methods 2.1. Materials l-[methyl-3H]carnitine and Sephadex G-25 (PD-10) were purchased from Amersham, MTS reagents from Toronto Research (North York, Ontario, Canada), Sephadex G-50, G-75 and G-200, egg-yolk phospholipids (l-αphosphatidylcholine from fresh turkey egg yolk), Pipes, Triton X-100, cardiolipin, l-carnitine and N-dodecanoylsarcosine (sarcosyl) from Sigma. All other reagents were of analytical grade.
2.2. Site-directed mutagenesis, overexpression and isolation of the CAC proteins The coding region for the rat CAC was amplified from total rat liver cDNA as described previously [1]. The Cys/Ser replacements were constructed with complementary mutagenic primers using the overlap extension method [24] and the High Fidelity PCR System (Roche). The PCR products were purified using the Gene Clean Kit (La Jolla), digested with NdeI and HindIII (restriction sites added at the 5′ end of forward and reverse primers, respectively) and ligated into the pMW7 expression vector. All mutations were verified by DNA sequencing and, except for the desired base changes, all of the sequences were identical to that of rat CAC cDNA. The resulting plasmids were transformed into Escherichia coli C0214 [15]. Bacterial overexpression, isolation of the inclusion body fraction, and solubilization and purification of the wild-type CAC and mutant CAC proteins were performed as described previously [15].
2.3. Reconstitution of CAC and CAC mutants in liposomes The recombinant proteins were reconstituted into liposomes as described previously [25]; however, the concentration of intraliposomal carnitine was varied as indicated in the legends to figures. The external substrate was removed from proteoliposomes on Sephadex G-75 columns.
2.4. Transport measurements Transport at 25 °C was started by adding [3H]carnitine, at the concentrations indicated in the legends to figures, to proteoliposomes and terminated by the addition of 1.5 mM NEM [15,25]. In the case of the mutant C23S/C89S/C136S/ C155S/C283S containing only Cys-58, the reaction was rapidly quenched by passing the samples through Sephadex G-50 at 2 °C. In controls, the inhibitor was added together with the labeled substrate, according to the inhibitor stop method [25]. Finally, the external substrate was removed by chromatography on Sephadex G-50 columns, and the radioactivity in the liposomes was measured. The initial transport rate was calculated from the radioactivity taken up by proteoliposomes after 1 min (in the initial linear range of substrate uptake). The experimental values were corrected by subtracting control values. All of the transport activities were determined by taking into account the efficiency of reconstitution (i.e., the share of successfully incorporated protein).
2.5. Other methods SDS-PAGE was performed according to Laemmli, as described previously [15]. The amount of recombinant protein was estimated on Coomassie Bluestained SDS-PAGE gels by the Bio-Rad GS-700 Imaging Densitometer equipped with the Bio-Rad Multi-Analist software using bovine serum albumin as standard. The extent of incorporation of the recombinant protein into liposomes was determined as described in Phelps et al. [26] with some modifications [15]. The homology model of the rat CAC was built based upon the structure of the bovine AAC1 [23] by using the computer application Swiss PDB Viewer [27]. A critical determinant in the accuracy of the comparative
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modeling is the quality of the pairwise alignment between the CAC and AAC1 template sequences. AAC1 and CAC were added to sequences from the mitochondrial carrier family and a multiple sequence alignment was generated by CLUSTALW. The secondary structure of AAC1 was used to weight the gap penalties as described previously [10]. One thousand steps of energy minimization were performed to generate 50 minimized CAC structures. The structural properties of the CAC models with the best energy function were evaluated using protein analysis tools available on the WHAT IF Web server [28]. Final models were examined with PyMOL, VMD and Swiss PDB Viewer and, where side-chain packing led to clashes, alternative side-chain rotamers were evaluated.
3. Results 3.1. Transport mechanism of CAC mutants The CAC contains six cysteines: Cys-23, Cys-58, Cys-89, Cys-136, Cys-155 and Cys-283. Wild-type CAC and CAC mutants, containing the combinations Cys-58/Cys-136, Cys-58/ Cys-155 and Cys-136/Cys-155 and the other four native cysteines replaced by serine, were overexpressed in E. coli, purified and reconstituted into liposomes to measure the transport rate of carnitine. In the comparative model of CAC,
Fig. 2. Bi-substrate analysis of the carnitine/carnitine antiport reaction catalyzed by reconstituted CAC mutants. Lineweaver–Burk plots showing the dependence of the carnitine/carnitine antiport rate on external carnitine concentration at three different internal carnitine concentrations. Transport was started by adding the indicated [3H]carnitine concentrations to liposomes reconstituted with the C58 + C136 mutant (A) or the C58 + C155 mutant (B) and preloaded internally with 2.5 mM (□), 7 mM (•) and 15 mM (○) unlabeled carnitine. Incubation time was 1 min. The Cys residues present in the mutants are shown. Similar results were obtained in three independent experiments in duplicate.
Fig. 1. Structural model of the CAC. Ribbon diagrams viewing the carrier from the lateral side. The transmembrane α-helices are colored as follows: H1, red; H2, tan (light brown); H3, green; H4, emerald-green; H5, cyan; and H6, blue. Purple surfaces highlight the SH groups of the Cys residues indicated by their number in the CAC sequence. Yellow surfaces highlight the salt bridge network between residues D32-K135, K35-D231 and E132-K234.
which was built using the structure of the ADP/ATP carrier in c-state as template [23], Cys-58, Cys-136 and Cys-155 are localised below the salt bridge network that closes the central pore of the α-helical bundle on the matrix side preventing translocation of the substrate (Fig. 1). The reconstituted CAC purified from rat liver mitochondria functions according to a ping-pong mechanism [14]. In this mechanism [29–32], only one substrate-binding site exists in the carrier, which is alternatively exposed to the external side (outward-facing conformation) or to the internal site (inwardfacing conformation) of the membrane. To ascertain whether the recombinant cysteine mutant carriers follow the same kinetic mechanism, the carnitine/carnitine exchange activity mediated by these proteins was measured by varying both external and internal substrate concentrations. When the kinetic data were analyzed in Lineweaver–Burk plots (Fig. 2A and B), showing the dependence of the transport rate on external carnitine at
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three different internal concentrations (2.5, 7.0 and 15.0 mM), a parallel pattern of straight lines was obtained with the CAC four Cys-replacement mutants C23S/C89S/C155S/C283S (containing Cys-58 and Cys-136) and C23S/C89S/C136S/C283S (containing Cys-58 and Cys-155). The external Km for carnitine of mutant C23S/C89S/C155S/C283S was increased from 0.4 to 2.4 mM (Fig. 2A) and that of the mutant C23S/C89S/C136S/ C283S from 0.1 to 1.0 mM (Fig. 2B) on increasing the internal substrate concentration from 2.5 to 15.0 mM. Clearly, variation of the substrate concentration in the internal compartment strongly influences the apparent transport affinity for the counter-substrate in the external compartment as a consequence of the change in the instant ratio between outward- and inwardfacing conformations of the carrier protein. Furthermore, secondary plots of the slopes and ordinate intercepts of Fig. 2A and B versus the reciprocal concentration of the substrate on the opposite side (not shown) demonstrated that the ratio of Km/Vmax did not change upon variation of the countersubstrate concentration. Similar results were obtained with the wild-type recombinant CAC and with the CAC C23S/C58S/ C89S/C283S mutant containing Cys-136 and Cys-155. Taken together these results show that all these proteins function according to a ping-pong mechanism. 3.2. Accessibility of Cys-58 to SH reagents To investigate the influence of the conformational state of the carrier on the accessibility of Cys-58, Cys-136 or Cys-155 to sulphydryl reagents, it would be ideal to use the five Cysreplacement mutants containing only Cys-58, Cys-136 or Cys155. However, all the CAC five Cys-replacement mutants had been previously found to be inactive [15]. Therefore, we first attempted to improve the activity of these mutants by adding increasing amounts of cardiolipin to the mixture used for their reconstitution into liposomes [11,33,34]. Fig. 3 shows that, although the wild-type CAC protein exhibited maximal activity at 200 μg/ml cardiolipin, the five replacement mutants C23S/ C89S/C136S/C155S/C283S, C23S/C89S/C58S/C155S/C283S and C23S/C58S/C89S/C136S/C283S (containing only Cys-58, Cys-136 and Cys-155, respectively) were virtually inactive at this cardiolipin concentration. At 500 μg/ml cardiolipin, however, the C23S/C89S/C136S/C155S/C283S mutant containing only Cys-58 displayed 40% transport activity compared to the wild-type CAC, whereas the other two 5 replacement mutants still exhibited negligible activity. Consequently, after its reconstitution in the presence of 500 μg/ml cardiolipin, the C23S/C89S/C136S/C155S/C283S mutant was tested for sensitivity to SH reagents. It was found that the carnitine transport activity of the mutant, measured as exchange between 0.1 mM external [3H]carnitine and 2.5 or 15.0 mM internal carnitine, was not affected by 0.5 mM MTSES or MTSEA, whether added outside the proteoliposomes even 10 min before adding the labeled substrate or present inside the proteoliposomes, i.e., during the reconstitution procedure (data not shown). Similarly, no effect of 0.5 mM MTSES or MTSEA on the C23S/C89S/ C136S/C155S/C283S mutant was observed when the activity was measured by adding 0.2 or 5 mM [3H]carnitine to pro-
Fig. 3. Dependence of CAC mutant activity on cardiolipin. Liposomes reconstituted with wild-type CAC (○), the C58 (•), C136 (▽) or C155 (▴) CAC mutant, in the presence of the indicated concentrations of cardiolipin, were preloaded internally with 13 mM carnitine. Transport was started with 0.1 mM [3H]carnitine and terminated after 10 min. Similar results were obtained in three independent experiments in duplicate.
teoliposomes containing 5 mM carnitine. In contrast, NEM inhibited the activity of the C23S/C89S/C136S/C155S/C283S mutant by about 80% at 0.5 mM when present in the internal compartment and had no effect up to 2.0 mM when preincubated externally for 10 min with the proteoliposomes (data not shown). These results indicate that Cys-58 is not accessible to MTSES and MTSEA, whereas it is accessible to NEM but only from the internal side of the reconstituted protein. 3.3. Accessibility of Cys-136 and Cys-155 to MTS reagents under variation of internal and external substrate concentrations Given that the five cysteine-replacement mutants C23S/ C89S/C58S/C155S/C283S and C23S/C58S/C89S/C136S/ C283S are inactive and Cys-58 is insensitive to externally added MTSES and MTSEA, the accessibility of Cys-136 and Cys-155 to these reagents was investigated in proteoliposomes reconstituted with one of the four Cys-replacement mutants, C23S/C89S/C155S/C283S, C23S/C89S/C136S/C283S and C23S/C58S/C89S/C283S, which contain the combinations Cys-58/Cys-136, Cys-58/Cys-155 and Cys-136/Cys-155, respectively. In a first set of experiments, MTSES or MTSEA was added together with 0.1 mM [3H]carnitine to proteoliposomes pre-loaded internally with two widely different carnitine concentrations (2.5 and 15.0 mM) and the initial carnitine/ carnitine exchange rate was measured. The residual transport activity in the presence of MTSES is shown in Fig. 4A–C. With all three mutants the inhibition of the transport rate by MTSES was clearly greater with 15.0 mM than with 2.5 mM internal carnitine. Furthermore, the mutant containing the Cys-58/Cys155 combination was much less sensitive to MTSES than the mutant containing the Cys-58/Cys-136 combination. Thus, half maximal inhibition of C23S/C89S/C136S/C283S was obtained
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mutants more in the presence of 15 mM than 2.5 mM internal carnitine and also inhibited more Cys-58/Cys-136 than Cys-58/ Cys-155 (data not shown). With the Cys-58/Cys-136 mutant, 4 μM MTSEA yielded total inhibition in the presence of 15 mM
Fig. 4. Effect of MTSES on the activity of C58 + C136, C58 + C155 and C136 + C155 mutants at different intraliposomal carnitine concentrations. Liposomes reconstituted with the C58 + C136 (A), C58 + C155 (B) or C136 + C155 (C) mutant were preloaded internally with 2.5 mM (○) or 15 mM (•) carnitine. Transport was started by adding 0.1 mM [3H]carnitine together with the indicated concentrations of MTSES and stopped after 1 min. Results are expressed as percent of residual activity (i.e., percent of activity in the presence of MTSES with respect to the control value without inhibitor). The data represent means ± SD of at least three independent experiments in duplicate. The Cys residues present in the CAC mutants are shown.
at a concentration more than one order of magnitude higher than that required for half maximal inhibition of C23S/C89S/C155S/ C283S. Similarly to MTSES, MTSEA inhibited the initial carnitine/carnitine exchange rate catalyzed by the same three
Fig. 5. Effect of MTSES on the activity of the C58 + C136, C58 + C155 and C136 + C155 mutants at different external carnitine concentrations. Liposomes were reconstituted with the C58 + C136 (A), C58 + C155 (B) or C136 + C155 (C) mutant and preloaded internally with 5 mM carnitine. Transport was started by adding 0.2 mM (□) or 5 mM (n) [3H]carnitine together with the indicated concentrations of MTSES. Incubation time was 1 min. Results are expressed as percent of residual activity (i.e., percent of activity in the presence of MTSES with respect to the control value without inhibitor). The data represent means ± SD of at least three independent experiments in duplicate. The Cys residues present in the CAC mutants are shown.
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internal carnitine and 8 μM MTSEA 93% inhibition in the presence of 2.5 mM internal carnitine; with the Cys-58/Cys-155 mutant, 8 μM MTSEA yielded 67% inhibition in the presence of 15 mM internal carnitine and 47% inhibition in the presence of 2.5 mM. All these results indicate that Cys-136 is more accessible than Cys-155 to MTSES and MTSEA and that the accessibility of both Cys residues is greater at the higher internal carnitine concentration, which favours the inward conformation of the protein. In other experiments, the effect of MTSES on the activity of the four Cys-replacement mutants C23S/C89S/C155S/C283S, C23S/C89S/C136S/C283S and C23S/C58S/C89S/C283S was measured in the presence of two different external carnitine concentrations (0.2 and 5.0 mM) at a constant internal concentration of 5.0 mM. MTSES inhibited all three mutants more with 0.2 mM than with 5.0 mM external carnitine (Fig. 5A–C). Results similar to those reported in Fig. 5 were obtained using MTSEA at concentrations up to 8 μM instead of MTSES (data not shown). Therefore, the accessibility of both Cys-136 and Cys-155 decreases upon increasing the external concentration, which favours the outward conformation of the carrier. Furthermore, as observed in the experiments where the substrate concentration was varied inside the proteoliposomes, the mutant containing the Cys-58/Cys-155 combination was less sensitive to MTSEA, and especially to MTSES, than the mutant containing the Cys-58/Cys-136 combination. It should be noted that the concentrations of substrate used outside and inside the
proteoliposomes in the experiments of Figs. 4 and 5 were chosen on the basis of the affinity of the carrier for carnitine on each side of the liposomal membrane [14,15] and on the fact that low internal and high external concentrations cannot be used simultaneously for reliable measurements of radioactive substrate uptake [25]. 3.4. Effect of intraliposomal carnitine concentration on Cys labeling by fluorescein-5-maleimide To further support the dependence of Cys-136 and Cys-155 accessibility on the intraliposomal substrate concentration, the effect of fluorescein-5-maleimide, another impermeable SH reagent [35], was investigated. In these experiments the mutants C23S/C58S/C89S/C283S, C23S/C89S/C155S/C283S and C23S/C89S/C136S/C283S containing Cys-136/Cys-155, Cys58/Cys-136 and Cys-58/Cys-155, respectively, were treated with fluorescein-5-maleimide in reconstituted liposomes under the two conditions of internal substrate concentration. Afterward, the mutant proteins were extracted, separated by SDSPAGE and either analyzed for fluorescence (Fig. 6A) or stained with silver nitrate (Fig. 6B). All three CAC mutants were more fluorescent when extracted from the proteoliposomes containing 15 mM internal carnitine than from the proteoliposomes containing 2.5 mM carnitine, indicating that the amount of bound fluorescein-5-maleimide depends on the internal carnitine concentration. In parallel experiments, reconstituted liposomes
Fig. 6. Influence of intraliposomal carnitine concentration on the labeling and inhibition of CAC mutants by fluorescein-5-maleimide. Liposomes reconstituted with the C136 + C155, C58 + C136 or C58 + C155 mutant and preloaded internally with 2.5 mM or 15 mM unlabeled carnitine were incubated with fluorescein-5maleimide, at 10 μM (A and B) and at the indicated concentrations (C) for 1 min at 25 °C; the reaction was then quenched by adding 10 mM DTE. The proteoliposomes were passed through Sephadex G-75 columns and ultracentrifuged at 110,000×g for 45 min at 4 °C; the pellets were suspended in 2% SDS, precipitated with acetone and subjected to SDS-PAGE. (A) Fluorescence of the UV-illuminated SDS-PAGE gel; (B) silver staining of the same gel; (C) inhibition of the carnitine/carnitine exchange activity by fluorescein-5-maleimide. Transport was started by adding 0.1 mM [3H]carnitine together with fluorescein-5-maleimide. Incubation time was 1 min. Results are expressed as percent of residual activity (i.e., percent of activity in the presence of fluorescein-5-maleimide with respect to the control value without inhibitor). The data represent means ± SD of three independent experiments in duplicate. The Cys residues present in the CAC mutants are shown. Abbreviation: FM, fluorescein-5-maleimide.
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treated with fluorescein-5-maleimide under the same conditions as in Fig. 6A were tested for residual transport activity (Fig. 6C). With all the mutants transport inhibition by fluorescein-5maleimide was increased by the presence of the higher intraliposomal concentration of carnitine, in agreement with the labeling results. Furthermore, virtually no labeling or inhibition by fluorescein-5-maleimide up to 10 μg/ml was detected in the case of the mutant C23S/C89S/C136S/C155S/C283S containing only Cys-58 reconstituted in the presence of 500 μg/ml cardiolipin (data not shown). 4. Discussion The CAC functions according to a ping-pong mechanism that implies the existence of two opposite conformations in which the substrate-binding site is alternatively exposed towards the cytosol (c-state or outward-facing conformation) or towards the matrix (m-state or inward-facing conformation). Unfortunately for CAC there are no side-specific ligands available that fix CAC in one of these conformations, as has been done with the ADP/ATP carrier by the addition of carboxyatractyloside or bongkrekic acid [17,36]. Here we took advantage of the transport mechanism of CAC, according to which the ratio between the inward- and the outward-facing conformation of the carrier can be varied by changing the concentration of the substrate inside or outside the proteoliposomes, in the presence of a fixed substrate concentration on the opposite side. Furthermore, the reconstituted CAC proteins were incubated with SH reagents only for the time interval used to measure the initial transport rate in order to limit the number of the catalytic cycles as much as possible. The experimental data reported above show that Cys-136 and Cys-155 of the reconstituted CAC become more accessible to the membrane-impermeable MTSES, MTSEA or fluorescein-5maleimide when the intraliposomal carnitine concentration is increased, therefore favouring the m-state of the carrier. Alternatively, these cysteines become less accessible when the concentration of carnitine is increased in the external compartment of the proteoliposomes, i. e., when the carrier is in the c-state. The three cysteines whose accessibility has been investigated here, Cys-58, Cys-136 and Cys-155, are located below the salt bridge network that in the c-state of the carrier closes the substrate translocation pathway on the matrix side (Fig. 1). Given that Cys-136 is located immediately below the salt bridge between K135 and D32 of the ionic network (Fig. 1), the greater accessibility of Cys-136 to externally added impermeable reagents when the majority of CAC molecules are in the mstate is most likely caused by the opening of the salt bridge network that must occur during the transition from the c- to the m-state. The conformational changes involved in substrate translocation are not yet known. However, it is thought that in the catalytic cycle of the ADP/ATP carrier the inter-repeat salt bridges between the charged residues of the three signature motifs break causing their rearrangement into intra-repeat salt bridges [37–39] and the stretching of the odd-numbered helices (H1, H3 and H5) [23] leading to the opening of the translocation
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channel. The corresponding inter-repeat salt bridges in rat CAC are D32-K135, E132-K234 and D231-K35, and the intra-repeat salt bridges are D32-K35, E132-K135 and D231-K234. The hypothesized straightening of the odd-numbered helices, in particular of H3, would make Cys-155 more accessible to externally added MTSES, MTSEA and fluorescein-5-maleimide in the m-state, although Cys-155 may be still partially screened by H3 in the m-state. This possibility may account for the lower sensitivity of Cys-155 toward methanethiosulfonate reagents compared to that of Cys-136. Furthermore, given that MTSES is negatively charged, the positive charge density of R133 and K135, that belong to H3 and are close to Cys-155, may diminish the interaction between MTSES and Cys-155. In a previous paper, it was shown that the reaction of externally added MTSES with Cys-136 of reconstituted CAC is prevented by the addition of carnitine to proteoliposomes [15]. It was suggested that Cys-136 is localised at or near the substratebinding site. Therefore, the substrate would impede the interaction of MTSES with Cys-136 either by direct competition with Cys-136 or by steric hindrance due to binding with side chains close to Cys-136. The results reported in this work, and in particular those obtained in the presence of different intraliposomal carnitine concentrations, favour the alternative explanation, i.e., that externally added substrate induces a conformational change of the carrier protein, thereby decreasing the binding of the inhibitors MTSES, MTSEA and fluorescein5-maleimide to a site different from that of the substrate. In this context it is worth mentioning that acetylcarnitine has been proposed to bind to F90, R180, D181 and R280 of the S. cerevisiae CAC [40], corresponding to the residues F86, R178, D179 and R275 of the rat CAC that are found in the cavity at the midpoint of the membrane. Unfortunately, it is not possible to ascertain whether Cys-136 is completely inaccessible in the cstate because the experimental approach used in this work does not allow to fix all of the carrier molecules in the same conformation. Furthermore, it should be stressed that the conformational change in the carrier can affect not only the accessibility but also the reactivity of Cys-136 and Cys-155. This may occur in different ways; for example, a change in the pK of the side chain of cysteine may take place when the microenvironment of the sulfhydryl group becomes more hydrophobic or hydrophilic. The results obtained using the CAC mutant containing only Cys-58 clearly show that Cys-58 behaves differently from Cys136 and Cys-155 because it is not accessible to MTSES, MTSEA and fluorescein-5-maleimide in all the experimental conditions used. This observation can be explained by the CAC homology model (Fig. 1), as Cys-58 is sterically hindered to externally added hydrophilic reagents being efficiently screened by H1. Unlike Cys-155, which is located at the beginning of h34, Cys-58 is located approximately at the midpoint of h12, and therefore the stretching of H1 occurring during the transition from the c-state to the m-state does not increase accessibility to Cys-58 as does the stretching of H3 to Cys-155. It could be argued that MTSES, MTSEA and fluorescein-5-maleimide bind Cys-58 without inhibiting the carrier. An argument in favour of the conclusion that they do not react with Cys-58 is the
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observation that when present inside the proteoliposomes, NEM inhibits the CAC mutant containing Cys-58 unlike Cys-less CAC. The accessibility of Cys-58 to NEM with respect to the hydrophilic reagents is favoured by the fact that this residue is surrounded by Y52, F59 and F64 that form an aromatic pocket. The results of the present study show that the matrix α-helix of the CAC that connects transmembrane helices H3 and H4, and contains Cys-136 and Cys-155, undergoes conformational changes during the catalytic cycle of the carrier. Our data are in line with the previous finding [21] that the corresponding region of the ADP/ATP carrier is accessible to externally added trypsin in a conformational-sensitive manner, being cleaved at the Lys178–Thr179 bond in the carrier-bongkrekic acid complex but not in the carrier-carboxyatractyloside complex. Indeed, positions 178 and 179 of the ADP/ATP carrier correspond to residues 157 and 158 of the CAC that are very close to Cys-155. This suggests rather similar conformational changes in the central matrix loop of the two mitochondrial carrier proteins. Acknowledgements This work was supported by grants from the Ministero dell'Università e della Ricerca (MUR), the Center of Excellence in Genomics (CEGBA), and EC contract LSHM-CT-2004503116. References [1] C. Indiveri, V. Iacobazzi, N. Giangregorio, F. Palmieri, The mitochondrial carnitine carrier protein: cDNA cloning, primary structure, and comparison with other mitochondrial transport proteins, Biochem. J. 321 (1997) 713–719. [2] V. Iacobazzi, M.A. Naglieri, C.A. Stanley, J.A.R. Wanders, F. Palmieri, The structure and organization of the human carnitine/acylcarnitine translocase (CACT) gene, Biochem. Biophys. Res. Commun. 252 (1998) 770–774. [3] L. Palmieri, F.M. Lasorsa, V. Iacobazzi, M.J. Runswick, F. Palmieri, J.E. Walker, Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae, FEBS Lett. 462 (1999) 472–476. [4] F. Palmieri, The mitochondrial transporter family (SLC25): physiological and pathological implications, in: M.A. Hediger (Ed.), The ABC of Solute Carriers, Pflugers Arch.-Eur. J. Physiol., vol. 447, 2004, pp. 689–709. [5] R. Krämer, F. Palmieri, in: L. Ernster (Ed.), Molecular Mechanisms in Bioenergetics, Elsevier Science Publishers, B.V., Amsterdam, 1992, pp. 359–384. [6] J.E. Walker, The mitochondrial transporter family, Curr. Opin. Struck. Biol. 2 (1992) 519–526. [7] E.R. Kunji, The role and structure of mitochondrial carriers, FEBS Lett. 564 (2004) 239–244. [8] N. Picault, M. Hodges, L. Palmieri, F. Palmieri, The growing family of mitochondrial carriers in Arabidopsis, Trends Plant Sci. 9 (2004) 138–146. [9] F. Palmieri, G. Agrimi, E. Blanco, A. Castegna, M.A. Di Noia, V. Iacobazzi, F.M. Lasorsa, C.M. Marobbio, L. Palmieri, P. Scarcia, S. Todisco, A. Vozza, J. Walker, Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins, Biochim. Biophys. Acta 1757 (2006) 1249–1262. [10] A.R. Cappello, R. Curcio, D.V. Miniero, I. Stipani, A.J. Robinson, E.R.S. Kunji, F. Palmieri, Functional and structural role of amino acid residues in the even-numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate carrier, J. Mol. Biol. 363 (2006) 51–62.
[11] C. Indiveri, A. Tonazzi, F. Palmieri, Identification and purification of the carnitine carrier from rat liver mitochondria, Biochim. Biophys. Acta 1020 (1990) 81–86. [12] A. Tonazzi, N. Giangregorio, C. Indiveri, F. Palmieri, Identification by site-directed mutagenesis and chemical modification of three vicinal cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter, J. Biol. Chem. 280 (2005) 19607–19612. [13] C. Indiveri, A. Tonazzi, F. Palmieri, Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria, Biochim. Biophys. Acta 1069 (1991) 110–116. [14] C. Indiveri, A. Tonazzi, F. Palmieri, The reconstituted carnitine carrier from rat liver mitochondria: evidence for a transport mechanism different from that of the other mitochondrial translocators, Biochim. Biophys. Acta 1189 (1994) 65–73. [15] C. Indiveri, N. Giangregorio, V. Iacobazzi, F. Palmieri, Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136, Biochemistry 41 (2002) 8649–8656. [16] M. Klingenberg, Molecular aspects of the adenine nucleotide carrier from mitochondria, Arch. Biochem. Biophys. 270 (1989) 1–14. [17] C. Fiore, V. Trezeguet, A. Le Saux, P. Roux, C. Schwimmer, A.C. Dianoux, F. Noel, G.J.-M. Lauquin, G. Brandolin, P.V. Vignais, The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects, Biochimie 80 (1998) 137–150. [18] E. Majima, K. Ikawa, M. Takeda, M. Hashimoto, Y. Shinohara, H. Terada, 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, J. Biol. Chem. 270 (1995) 29548–29554. [19] M. Hashimoto, E. Majima, S. Goto, Y. Shinohara, H. Terada, Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides, Biochemistry 38 (1999) 1050–1056. [20] Y. Kihira, A. Iwahashi, E. Majima, H. Terada, Y. Shinohara, Twisting of the second transmembrane α-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states, Biochemistry 43 (2004) 15204–15209. [21] C. Dahout-Gonzalez, C. Ramus, E.P. Dassa, A.-C. Dianoux, G. Brandolin, Conformation-dependent swinging of the matrix loop m2 of the mitochondrial Saccharomyces cerevisiae ADP/ATP carrier, Biochemistry 44 (2005) 16310–16320. [22] A. Iwahashi, Y. Kihira, E. Majima, H. Terada, N. Yamazaki, M. Kataoka, Y. Shinohara, The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state, Mitochondrion 6 (2006) 245–251. [23] E. Pebay-Peyroula, C. Dahout-Gonzalez, R. Kahn, V. Trezeguet, G.J. Lauquin, G. Brandolin, Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside, Nature 426 (2003) 39–44. [24] S.N. Ho, H.D. Hunt, R.M. Horton, J.K. Pullen, L.R. Pease, Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77 (1989) 51–59. [25] F. Palmieri, C. Indiveri, F. Bisaccia, V. Iacobazzi, Methods Enzymol. 260 (1995) 349–369. [26] A. Phelps, C. Briggs, L. Mincone, H. Wohlrab, Mitochondrial phosphate transport protein. Replacements of glutamic, aspartic, and histidine residues affect transport and protein conformation and point to a coupled proton transport path, Biochemistry 35 (1996) 10757–10762. [27] N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling, Electrophoresis 18 (1997) 2714–2723. [28] G. Vriend, WHAT IF: a molecular modeling and drug design program, J. Mol. Graph. 8 (1990) 52–56. [29] W.W. Cleland, in: P.D. Boyer (Ed.), The Enzymes, vol. 2, Academic Press, New York, 1970, pp. 1–65. [30] I.H. Segel, Enzyme kinetics, Wiley, New York, 1975, pp. 274–320. [31] W. Furuya, T. Tarshis, F.-Y. Law, P.A. Knauf, J. Gen. Physiol. 83 (1984) 657–681.
N. Giangregorio et al. / Biochimica et Biophysica Acta 1767 (2007) 1331–1339 [32] F. Palmieri, M. Klingenberg, Mitochondrial metabolite transporter family, in: W.J. Lennarz, M.D. Lane (Eds.), Encyclopedia of Biological Chemistry, vol. 2, Elsevier, Oxford, 2004, pp. 725–732. [33] G. Fiermonte, V. Dolce, F. Palmieri, Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria, J. Biol. Chem. 273 (1998) 22782–22787. [34] R.S. Kaplan, J.A. Mayor, D. Brauer, R. Kotaria, D.D. Walters, A.M. Dean, The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway, J. Biol. Chem. 275 (2000) 12009–12016. [35] P.C. Jones, A. Sivaprasadarao, D. Wray, J.B. Findlay, A method for determining transmembrane protein structure, Mol. Membr. Biol. 13 (1996) 53–60.
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[36] M. Klingenberg, Dialectics in carrier research: the ADP/ATP carrier and the uncoupling protein, J. Bioenerg. Biomembranes 25 (1993) 447–457. [37] D.R. Nelson, C.M. Felix, J.M. Swanson, Highly conserved charge-pair networks in the mitochondrial carrier family, J. Mol. Biol. 277 (1998) 285–308. [38] E.R. Kunji, A.J. Robinson, The conserved substrate binding site of mitochondrial carriers, Biochim. Biophys. Acta 1757 (2006) 1237–1248. [39] M. Falconi, G. Chillemi, D. Di Marino, I. D'Annessa, B. Morozzo della Rocca, L. Palmieri, A. Desideri, Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies, Proteins 65 (2006) 681–691. [40] A.J. Robinson, E.R. Kunji, Mitochondrial carriers in the cytoplasmic state have a common substrate binding site, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 2617–2622.