Identification by site-directed mutagenesis and chemical modification of three vicinal cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter

The proximity of the Cys residues present in the mitochondrial rat carnitine/acylcarnitine carrier (CAC) primary structure was studied by using site-directed mutagenesis in combination with chemical modification. CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli and reconstituted into liposomes. The effect of SH oxidizing, cross-linking, and coordinating reagents was evaluated on the carnitine/carnitine exchange catalyzed by the recombinant reconstituted CAC proteins. All the tested reagents efficiently inhibited the wild-type CAC. The inhibitory effect of diamide, Cu(2+)-phenanthroline, or phenylarsine oxide was largely reduced or abolished by the double substitutions C136S/C155S, C58S/C136S, and C58S/C155S. The decrease in sensitivity to these reagents was much lower in double mutants in which Cys(23) was substituted with Cys(136) or Cys(155). No decrease in inhibition was found when Cys(89) and/or Cys(283) were replaced with Ser. Sb(3+), which coordinates three cysteines, inhibited only the Cys replacement mutants containing cysteines 58, 136, and 155 of the six native cysteines. In addition, the mutant C23S/C89S/C155S/C283S, in which double tandem fXa recognition sites were inserted in positions 65-72, i.e. between Cys(58) and Cys(136), was not cleaved into two fragments by fXa protease after treatment with diamide. These results are interpreted in light of the homology model of CAC based on the available x-ray structure of the ADP/ATP carrier. They indicate that Cys(58), Cys(136), and Cys(155) become close in the tertiary structure of the CAC during its catalytic cycle.     

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

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-maleimide-treated 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.     

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

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-maleimide-treated 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.     

Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: an inhibition kinetic approach combined with the analysis of the homology structural model

To gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH(2) residues of the active site should be comparable to the distance between the gamma-NH(2) and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue.     

Glutathione controls the redox state of the mitochondrial carnitine/acylcarnitine carrier Cys residues by glutathionylation

Background

The mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches.

Methods

The effect of GSH and GSSG on the [³H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody.

Results

GSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37 °C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC.

Conclusions

CAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation.

General significance

CAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations.     

Site-directed sulfhydryl labeling of helix IX in the lactose permease of Escherichia coli

Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-[(14)C]ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively. Out of approximately 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM. (i) Cys residues at positions 291, 308, and 310 label at 25 degrees C, and binding of substrate has no effect. (ii) Cys residues at positions 295 and 298 label only in the presence of substrate. NEM labeling at 0 degrees C indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein. In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310. Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants. The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane. Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover [Sahin-Tóth, M., and Kaback, H. R. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6068-6073], the findings are consistent with the idea that this face of helix IX may comprise part of the H(+) translocation pathway.     

The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter

The sucrose (CscB) permease belongs to the oligosaccharide:H(+) symporter family of the Major Facilitator Superfamily and is homologous to the lactose permease from Escherichia coli. Sucrose transport in cells expressing sucrose permease is completely inhibited by N-ethylmaleimide (NEM), suggesting that one or more of the seven native Cys residues may be important for transport. In this paper, each Cys residue was individually replaced with Ser, and transport activity, membrane expression, and NEM sensitivity are documented. All seven single Cys-->Ser mutants are expressed normally in the membrane and catalyze sucrose transport with activities ranging from 80% to 180% of wild type. Six of the seven Ser mutants are completely inactivated by NEM, while Cys122-->Ser permease is insensitive to the sulfhydryl reagent, indicating that NEM inhibition results from alkylation of Cys122. Subsequently, a sucrose permease devoid of Cys residues (Cys-less) was constructed in which all Cys residues were replaced with Ser simultaneously by using a series of overlap-extension PCRs. Membrane expression and kinetic parameters for Cys-less [K(m) 4.8 mM, V(max) 192 nmol min(-1) (mg of protein)(-1)] are essentially identical to those of wild type [K(m) 5.4 mM, V(max) 196 nmol min(-1) (mg of protein)(-1)]. However, Cys-less permease catalyzes sucrose accumulation to steady-state levels that are approximately 2-fold higher than those of wild type. As anticipated, Cys-less permease is completely resistant to NEM inhibition. The observations demonstrate that Cys residues play no functional role in sucrose permease. Furthermore, the approach described to create the Cys-less transporter is generally applicable to other proteins. An application of Cys-less permease in the study of the substrate binding site is presented in the accompanying paper.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: N-ethylmaleimide-sensitive face of helix II

Cys-scanning mutagenesis of helix II in the lactose permease of Escherichia coli [Frillingos, S., Sun, J. et al. (1997) Biochemistry 36, 269-273] indicates that one face contains positions where Cys replacement or Cys replacement followed by treatment with N-ethylmaleimide (NEM) significantly inactivates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [(14)C]NEM labeling of 13 single-Cys mutants, including the nine NEM-sensitive Cys replacements, in right-side-out membrane vesicles is examined. Permease mutants with a single-Cys residue in place of Gly46, Phe49, Gln60, Ser67, or Leu70 are alkylated by NEM at 25 degrees C in 10 min, and mutants with Cys in place of Thr45 and Ser53 are labeled only in the presence of ligand, while mutants with Cys in place of Ile52, Ser56, Leu57, Leu62, Phe63, or Leu65 do not react. Binding of substrate leads to a marked increase in labeling of Cys residues at positions 45, 49, or 53 in the periplasmic half of helix II and a slight decrease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) show that positions 45 and 53 are accessible to solvent in the presence of ligand only, while positions 46, 49, 67, and 70 are accessible to solvent in the absence or presence of ligand. Position 60 is also exposed to solvent, and substrate binding causes a decrease in solvent accessibility. The findings demonstrate that the NEM-sensitive face of helix II participates in ligand-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membrane. In the following paper in this issue [Venkatesan, P., Hu, Y., and Kaback, H. R. (2000) Biochemistry 39, 10656-10661], the approach is applied to helix X.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na+, with a positive cooperativity. Na+ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes.

EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cys-less) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway.     

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.     

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.     

Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential

Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced.     

Reconstitution into liposomes and functional characterization of the carnitine transporter from renal cell plasma membrane

The carnitine transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalysed a first-order antiport reaction (carnitine/carnitine or carnitine/substrate) stimulated by external, not internal, Na⁺, with a positive cooperativity. Na⁺ was co-transported with carnitine. Optimal activity was found between pH 5.5 and pH 6.0. The sulfhydryl reagents MTSES, MTSET and mercurials strongly inhibited the transport. Substrate analogues inhibited the transport; the most effective were acylcarnitines and betaine, followed by dimethylglicine, tetraethylammonium and arginine. Besides carnitine, only acylcarnitines and betaine were efficiently translocated. The Km for carnitine on the external and internal side of the transporter was 0.08 and 1.2 mM, respectively. The transporter is asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. The reconstituted carnitine transporter corresponds, very probably, to the OCTN2 protein.     

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.     

Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif

The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane alpha-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids.     

The yeast mitochondrial citrate transport protein. Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.     

Site-directed alkylation of cysteine replacements in the lactose permease of Escherichia coli: helices I, III, VI, and XI

To complete a study on site-directed alkylation of Cys replacements in the lactose permease of Escherichia coli (LacY), the reactivity of single-Cys mutants in helices I, III, VI, and XI, as well as some of the adjoining loops, with N-[14C]ethylmaleimide (NEM) or methanethiosulfonate ethylsulfonate (MTSES) was studied in right-side-out membrane vesicles. With the exception of several positions in the middle of helix I, which either face the bilayer or are in close proximity to other helices, the remaining Cys replacements react with the membrane-permeant alkylating agent NEM. In helices III and XI, most Cys replacements are also alkylated by NEM except for positions that face the bilayer. The reactivity of Cys replacements in helix VI is noticeably lower and only 45% of the replacements label. Binding of sugar leads to significant increases in the reactivity of Cys residues that are located primarily at the same level as the sugar-binding site or in the periplasmic half of each helix. Remarkably, studies with small, impermeant MTSES show that single-Cys replacements in the cytoplasmic portions of helices I and XI, which line the inward-facing cavity, are accessible to solvent from the periplasmic surface of the membrane. Moreover, addition of ligand results in increased accessibility of Cys residues to the aqueous milieu in the periplasmic region of the helices, which may reflect structural rearrangements leading to opening of an outward-facing cavity. The findings are consistent with the X-ray structure of LacY and with the alternating access model [Abramson, J., Smirnova, I., et al. (2003) Science 301, 610-615].     

Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif

The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids.     

Citrate uniport by the mitochondrial tricarboxylate carrier: a basis for a new hypothesis for the transport mechanism

The tricarboxylate transport system located in the inner mitochondrial membrane was studied as an isolated protein reconstituted in proteoliposomes. The effects on the transport of citrate by various reagents, specific for different aminoacid residues, were analyzed. In the group of SH reagents, it was found that N-ethylmaleimide is an irreversible inhibitor of the citrate–citrate exchange, while HgCl₂ and the mercurial mersalyl cause a rapid unidirectional efflux of citrate from liposomes. It was demonstrated that NEM and mercurials act on different SH groups. Dithioerythritol is not able to reverse the effect of mersalyl unless another reagent, pyridoxalphosphate, is present. Pyridoxalphosphate itself, a reagent specific for NH₂ residues, is an effective inhibitor of citrate exchange transport, as measured in both influx and efflux, but it has no effect on the mercurial-induced efflux. The same behavior was observed with diethylpyrocarbonate, a reagent specific for histidine and tyrosine residues. Interestingly, a slow basic efflux of internal citrate, in the absence of countersubstrate, was observed in proteoliposomes. Because it is inhibited by the same reagents acting on the exchange process, it is deduced that it is catalyzed by the tricarboxylate carrier. The ability of the carrier to perform a uniport of the substrate suggests the presence of a single substrate binding site on the carrier protein. A preliminary kinetic approach indicates that such a transport model is compatible with this theory.