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• W2039717152 abstract "The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine. A cysteine-less CTP that retains wild-type functional properties, served as the starting template. The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function. These rate constants varied by over five orders of magnitude. With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137. Based on the pattern of accessibility we conclude that residues 123-137 exist as an α-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the α-helical secondary structure may extend to residue 113. Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII′ dimer interface has been developed. The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine. A cysteine-less CTP that retains wild-type functional properties, served as the starting template. The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function. These rate constants varied by over five orders of magnitude. With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137. Based on the pattern of accessibility we conclude that residues 123-137 exist as an α-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the α-helical secondary structure may extend to residue 113. Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII′ dimer interface has been developed. The mitochondrial citrate transport protein (i.e. CTP) 1The abbreviations used are: CTP, citrate transport protein; BTC, 1,2,3-benzenetricarboxylate; EPR, electron paramagnetic resonance; MTS, methanethiosulfonate; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; MTSET, (2-(trimethylammonium)ethyl)methanethiosulfonate bromide; TMD, transmembrane domain. from higher eukaryotic cells catalyzes an electroneutral exchange across the inner mitochondrial membrane of a tricarboxylate (i.e. citrate, isocitrate, and cis-aconitate) plus a proton, for either another tricarboxylate-H+, a dicarboxylate (i.e. malate or succinate), or phosphoenolpyruvate (1.Palmieri F. Stipani I. Quagliariello E. Klingenberg M. Eur. J. Biochem. 1972; 26: 587-594Crossref PubMed Scopus (173) Google Scholar). The CTP occupies a critical position in intermediary metabolism, because the resulting cytoplasmic citrate serves as a carbon source, which fuels both the fatty acid and the sterol biosynthetic pathways, and provides a source of NAD+ (via the concerted actions of ATP-citrate lyase and malate dehydrogenase) for the glycolytic pathway (2.Watson J.A. Lowenstein J.M. J. Biol. Chem. 1970; 245: 5993-6002Abstract Full Text PDF PubMed Google Scholar, 3.Endemann G. Goetz P.G. Edmond J. Brunengraber H. J. Biol. Chem. 1982; 257: 3434-3440Abstract Full Text PDF PubMed Google Scholar, 4.Brunengraber H. Lowenstein J.M. FEBS Lett. 1973; 36: 130-132Crossref PubMed Scopus (37) Google Scholar, 5.Conover T.E. Trends Biochem. Sci. 1987; 12: 88-89Abstract Full Text PDF Scopus (35) Google Scholar). Due to its prominent role, the CTP has been intensively studied. Thus, it has been purified (6.Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar, 7.Bisaccia F. De Palma A. Palmieri F. Biochim. Biophys. Acta. 1989; 977: 171-176Crossref PubMed Scopus (102) Google Scholar), kinetically characterized (8.Bisaccia F. De Palma A. Prezioso G. Palmieri F. Biochim. Biophys. Acta. 1990; 1019: 250-256Crossref PubMed Scopus (58) Google Scholar), cloned (9.Kaplan R.S. Mayor J.A. Wood D.O. J. Biol. Chem. 1993; 268: 13682-13690Abstract Full Text PDF PubMed Google Scholar), and overexpressed (10.Xu Y. Mayor J.A. Gremse D. Wood D.O. Kaplan R.S. Biochem. Biophys. Res. Commun. 1995; 207: 783-789Crossref PubMed Scopus (45) Google Scholar). More recently, our investigations of this transporter have focused on the yeast homologue of the higher eukaryotic protein (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). An advantage provided by the yeast mitochondrial CTP is that, following overexpression and subsequent purification, CTP function can be reconstituted in liposomal vesicles with high specific activity. Thus, the yeast CTP represents ideal material with which to carry out detailed structure/function studies. Accordingly, we have developed a Cys-less CTP construct that displays near-native functional properties (12.Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and have demonstrated that, upon isolation, both the wild-type and Cys-less CTPs exist as functional dimers (13.Kotaria R. Mayor J.A. Walters D.E. Kaplan R.S. J. Bioenerg. Biomembr. 1999; 31: 543-549Crossref PubMed Scopus (38) Google Scholar). Based on hydropathy analysis, each monomer of the homodimer is predicted to contain six membrane-spanning domains (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). With this background in mind, we have begun to investigate each of the CTP transmembrane domains with the goal of elucidating their secondary structure and identifying their water-accessible and -inaccessible surfaces. To date, we have carried out this type of analysis with TMDIV (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Within the CTP, transmembrane domain III (TMDIII) appears to be of particular importance to CTP structure and function for several reasons. First, two negatively charged residues exist within this TMD (Glu122 and Glu131), as well as one or possibly two positively charged residues (Arg110 and Lys134) depending on the exact location of the membrane/aqueous compartment interface. Interestingly, with the exception of Arg110, each of these charged residues is conserved among CTPs of widely divergent origin (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Second, the amino-terminal segment of TMDIII contains the dimerization consensus sequence 115GXXXG119, which is thought to be involved in formation of a high affinity association between transmembrane domains (15.MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (873) Google Scholar, 16.Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (785) Google Scholar, 17.Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (515) Google Scholar, 18.Fleming K.G. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14340-14344Crossref PubMed Scopus (158) Google Scholar, 19.Liu, Y., Engelman, D. M., and Gerstein, M. (2002) Genome Biology http://genomebiology.com/2002/3/10/research/0054Google Scholar) and is conserved among CTPs (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Importantly, we have demonstrated by two different approaches that the CTP exists as a homodimer in solution (13.Kotaria R. Mayor J.A. Walters D.E. Kaplan R.S. J. Bioenerg. Biomembr. 1999; 31: 543-549Crossref PubMed Scopus (38) Google Scholar). Finally, our molecular modeling studies suggest the possibility that TMDIII of each CTP monomer may not only form part of the dimer interface, but might also contribute residues to the translocation pathway present in each CTP monomer (20.Walters D.E. Kaplan R.S. J. Mol. Model. 2000; 6: 587-594Crossref Google Scholar). In combination, the above findings point to the importance of this transmembrane domain in CTP function. Here, we report findings from experiments designed to investigate the topology of TMDIII. Utilizing the Cys-less CTP as the starting template, we mutated one at a time, 28 consecutive residues within TMDIII to cysteine. We then overexpressed, solubilized, and functionally reconstituted each single-Cys mutant in liposomal vesicles and measured the accessibility of each mutant to the hydrophilic, cysteine-specific reagents MTSES and MTSET by determining the rate constant for inhibition of citrate transport. Depending upon the location of a given Cys in the primary structure, these rate constants differed by over five orders of magnitude. Based on the periodicity of the accessibility of residues 112-137 to both MTS derivatives, our results clearly show that TMDIII exists as an α-helix from residues 123-137. Furthermore, our data suggest that this secondary structure may extend to residue 113. Importantly, both the water-accessible and -inaccessible faces of this helix have been clearly delineated. We infer from these investigations that the water-accessible face represents a portion of the substrate translocation pathway through the CTP, and the water-inaccessible surface faces the membranous lipid bilayer. Finally, a model for a putative TMDIII-TMDIII′ dimer interface has been proposed. Construction, Overexpression, and Isolation of Single-Cys CTP Variants—Single-Cys CTP variants were prepared employing the QuikChange site-directed mutagenesis kit (Stratagene). Primers containing the desired mutation were analyzed with Oligo 5.0 software and were purchased commercially. PCR amplifications were conducted utilizing the Cys-less CTP gene in pET-21a(+) (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) as the starting template. Amplification reactions and the subsequent cloning steps were performed according to the manufacturer's instructions. Transformants were screened for the presence of inserts via restriction digestion of purified plasmid DNA with NdeI and BamHI. The DNA from positive clones was then partially sequenced to ensure the presence of the desired mutation. Plasmids scoring positively for the appropriate mutation were subcloned into the storage (NovaBlue) and expression (BL21(DE3)) hosts as previously described (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Mutations were confirmed by sequencing both strands of the entire CTP open reading frame using plasmid, purified from the expression strain, as the template. DNA sequencing was performed by the fee-for-service Iowa State University DNA Sequencing and Synthesis Facility. High level expression of single-Cys CTPs in Escherichia coli, isolation of the inclusion body fraction, and solubilization of the CTP mutants from inclusion bodies with 1.2% Sarkosyl were performed as previously discussed (10.Xu Y. Mayor J.A. Gremse D. Wood D.O. Kaplan R.S. Biochem. Biophys. Res. Commun. 1995; 207: 783-789Crossref PubMed Scopus (45) Google Scholar, 11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Incorporation of Single-Cys CTP Variants into Phospholipid Vesicles and Determination of the Quantity of Incorporated Protein—Overexpressed, Sarkosyl-solubilized, single-cys CTP mutants were incorporated into phospholipid vesicles via the freeze-thaw-sonication procedure as previously described (6.Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar, 11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Immediately prior to the transport assay, a given sample was thawed, sonicated on ice (30 sonic bursts; 0.7 s/burst), and extraliposomal citrate was removed on small Dowex columns. The sample was then immediately assayed for transport. Where indicated, the quantity of protein that actually incorporated into phospholipid vesicles was determined via the method of Kaplan and Pedersen (21.Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar) as previously detailed (6.Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar, 14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Measurement of the Effects of MTSES and MTSET on Reconstituted Citrate Transport—The effects of the MTS reagents on BTC-sensitive reconstituted citrate transport was determined as previously described (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Briefly, proteoliposomes (45 μl) were preincubated with buffer (3.5 μl) (6.Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. J. Biol. Chem. 1990; 265: 13379-13385Abstract Full Text PDF PubMed Google Scholar) for 30 s followed by the addition of freshly prepared MTS reagent (3.5 μl). The concentration of MTS reagent utilized was determined by an initial series of experiments with each CTP variant, in which the MTSET concentration was varied by several orders of magnitude to determine both the maximal inhibition that could be obtained, as well as the levels of reagent needed to obtain varying extents of inhibition at rates that could be measured (i.e. in the range of 5 s to 5 h). Following preincubation with a given concentration of MTS reagent, transport was subsequently triggered via the addition of 21.5 μl of [1,5-14C]citrate (Amersham Biosciences; specific radioactivity approximately 2.4 × 104 dpm/nmol; concentration in reaction mix = 1.04 mm). The incubation time was determined based on the intrinsic activity of a given mutant. These reaction times were empirically determined such that in the absence of MTS reagent less than ∼2500 cpm were transported into the liposomes in a BTC-sensitive manner. We have previously demonstrated that these conditions approximate initial rate conditions (12.Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Transport incubations, which were carried out at 21 °C, were quenched via the addition of 3.5 μl of BTC (final concentration in reaction mix = 9.0 mm). Employing the above reaction sequence, multiple reactions were carried out in which the length of the preincubation with the MTS reagent prior to the citrate addition was varied, thereby generating a time course for the extent of inactivation. Experimental and control incubations were carried out during each time course experiment. With these incubations, water was added instead of the MTS reagent. Otherwise the experimental transport reaction was carried out exactly as described above, except only a single time interval between the water addition and the addition of [14C]citrate was utilized. For the control incubation the order of addition of buffer and BTC was reversed. Thus the control received 3.5 μl of BTC at the beginning of the preincubation sequence and buffer instead of BTC at the end of the reaction sequence. Therefore, the experimental and control tubes received the identical concentrations of each component, albeit with the order of additions being altered. Finally, it should be noted that with the Gly115 → Cys and Ala118 → Cys mutations, incomplete inhibition of the CTP by BTC was observed. Therefore, mersalyl was substituted for BTC in the MTSET studies. Following all incubations, intraliposomal radiolabeled citrate was separated by chromatography on small Dowex columns (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and was quantified via liquid scintillation counting. The proportion of activity remaining after incubation with a given MTS reagent was determined by: (i) subtracting the control transport rate from the rate observed in the presence of MTS reagent and (ii) determining the ratio of this difference to the uninhibited BTC-sensitive transport rate (i.e. the experimental rate - control rate measured in the presence of water instead of MTS reagent). Finally, control studies, using liposomes reconstituted with the Cys-less CTP, indicated that the observed effects of MTSES and MTSET were not due to a nonspecific effect on either the liposomal bilayer or the CTP. Calculation of the Rate Constants for MTS Inactivation of CTP Function—Time-course data for inactivation of the CTP by MTSES and MTSET were fitted to a simple exponential function by unweighted Marquardt nonlinear least squares according to the following equation: rt = (r0 - r∞)·e-m·c·t + r∞, where rt is the observed activity remaining at time t (seconds), r0 is the initial activity, r∞ is the asymptotic activity at t = ∞, c is reagent concentration (μm-1), and m is the rate constant for inactivation (s-1 μm-1) (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Plots of the log10 transformed estimates of m against residue number revealed simple linear trends in the data that were fitted by least squares. The superimposed sinusoids were manually fitted to the data. Overall Strategy—Based on the reasons articulated in the introduction, TMDIII appears to play one or more particularly important roles in the structure and function of the CTP. Accordingly, we utilized the following approach to investigate its topology. First, we mutated residues 110-137 one at a time to cysteine, using a Cys-less CTP as the starting template. As shown in Fig. 1, these residues are thought to comprise TMDIII. Second, we overexpressed each of the 28 single-Cys CTP mutants, as well as the starting Cys-less CTP, in E. coli. Each CTP variant was extracted from isolated inclusion bodies with the anionic detergent Sarkosyl and then functionally reconstituted in liposomes. We have previously demonstrated that this strategy (11.Kaplan R.S. Mayor J.A. Gremse D.A. Wood D.O. J. Biol. Chem. 1995; 270: 4108-4114Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 12.Xu Y. Kakhniashvili D.A. Gremse D.A. Wood D.O. Mayor J.A. Walters D.E. Kaplan R.S. J. Biol. Chem. 2000; 275: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) yields abundant quantities of the wild-type and the Cys-less CTPs, which are highly functional when incorporated into phospholipid vesicles (i.e. they display native kinetic properties and substrate specificity). Third, we characterized the ability of the reagents MTSES and MTSET to inhibit the function of each CTP mutant. Thus, with each single-Cys CTP mutant the rate constant for inhibition by each MTS reagent was determined. There are several suppositions implicit to this approach, which have been previously discussed in detail (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22.Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 23.Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24.Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 25.Kurz L.L. Zuhlke R.D. Zhang H.-J. Joho R.H. Biophys. J. 1995; 68: 900-905Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 26.Javitch J.A. Fu D. Chen J. Karlin A. Neuron. 1995; 14: 825-831Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 27.Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar), and thus will be mentioned here only briefly. First, it is postulated that if, following mutation of a given wild-type residue to Cys, significant citrate transport is retained, then the structure of the mutated transporter, including the location of the introduced Cys in the transporter's three-dimensional structure, is not substantively changed from that of the wild-type carrier. Second, it is postulated that, within a transmembrane domain, only those amino acids that reside on a water-accessible surface of the transporter will rapidly react with MTSES and MTSET. This idea is founded on the observations that: (i) these two polar reagents are impermeable to a lipid bilayer in the absence of a protein-mediated channel or translocation pathway (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22.Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 28.Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996; 35: 797-804Crossref PubMed Scopus (193) Google Scholar) and thus will not readily access residues facing the hydrophobic bilayer and (ii) MTS derivatives react >109 more rapidly with the thiolate anion (formed only upon exposure of the sulfhydryl group to water) compared with the unionized thiol group (27.Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 29.Roberts D.D. Lewis S.D. Ballou D.P. Olson S.T. Shafer J.A. Biochemistry. 1986; 25: 5595-5601Crossref PubMed Scopus (200) Google Scholar). Third, it is assumed that the covalent addition of -SCH2CH2X to a cysteine will be sufficiently disruptive to inhibit transport. Thus, rapid inhibition of transport is interpreted as evidence that the modified Cys side chain is located within the aqueous transport pathway in a domain with sufficiently narrow dimensions such that the modification blocks substrate transport. Lastly, because citrate is hydrophilic, of similar size and occupies a volume similar to that of the MTS reagents (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), we postulate that both substrate and the MTS reagents will access similar domains and thus the MTS-accessible surface of a transmembrane domain constitutes a portion of the citrate translocation pathway through the CTP. Functional Characterization of CTP Single-cysteine Replacement Mutants—Fig. 2A depicts the reconstituted citrate transport specific activity values observed with the Cys-less CTP and the 28 single-Cys CTP variants. It is noteworthy that, although all of the single Cys mutants displayed measurable BTC-sensitive citrate transport activity, they could be divided into three categories, based on their sensitivity to Cys replacement. The first category consists of five mutants (G119C, E122C, S123C, E131C, and K134C) that displayed severe functional defects such that they retained less than 4% of the starting Cys-less function, thereby suggesting that the wild-type residues at these positions are extremely intolerant of Cys substitution and may be essential to CTP function. The second category consists of three mutants (G115C, V127C, and I133C) that displayed significant, but nonetheless substantially impaired function, such that >4% and ≥16% of the initial Cys-less function was retained. The third category consists of the remaining 20 mutants that were either functionally unaffected or were only mildly impaired. These mutants displayed specific activity values that ranged from 32 to 135% of the starting Cys-less CTP, with a mean value of 74%. Interestingly, the 8 residues that are extremely sensitive to mutation (i.e. Categories 1 and 2; a mean of 5.8% of the starting Cys-less activity was retained) occur with an average periodicity of 3.4 residues and therefore likely reside on one face of an α-helix. These residues are colored red in Fig. 2B. Mutation of the remaining 20 residues on other surfaces of the helix are considerably less disruptive of CTP function, as evidenced by the fact that a mean specific activity value of 74% of the initial Cys-less activity was retained. These residues are denoted in blue in Fig. 2B. Finally, because the results presented in Fig. 2A indicate that, with only a few exceptions, most of the single-Cys CTP variants displayed significant citrate transport activity, we conclude that the overall structural and functional integrity of the CTP has been preserved with this panel of mutants. Thus, an important prerequisite for initiation of the strategy of probing single Cys accessibilities to MTS reagents has been met, and this approach is likely to yield important structural information (25.Kurz L.L. Zuhlke R.D. Zhang H.-J. Joho R.H. Biophys. J. 1995; 68: 900-905Abstract Full Text PDF PubMed Scopus (99) Google Scholar). Two further points should be noted. First, with each single Cys CTP mutant, we directly measured the proportion of added protein that actually incorporated into the liposomal vesicles (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We observed with the 28 single Cys mutants that 69.3% ± 5.9% (mean ± S.D. for the 28 mutants) of added protein incorporated into the liposomes. Thus we conclude that each of the mutated CTPs incorporated into phospholipid vesicles to a similar degree and that the alterations observed in reconstituted citrate transport activity (i.e. Fig. 2) are not a consequence of preferential incorporation of one mutant versus another. Second, a high degree of bacterial overexpression was achieved with each of the single Cys CTP mutants. Thus, upon solubilization of inclusion body protein with the detergent Sarkosyl, we obtained a mean value of 60 mg of each single-Cys CTP variant per liter of E. coli culture (range = 25-88 mg/liter) at a purity of ∼70%. Accessibility of Single Cys CTP Mutants to MTSES- and MTSET+—We measured the accessibility of each single Cys CTP mutant to MTSES and MTSET. These reagents are small, water-soluble, cysteine-specific, and display opposite charges (22.Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar). They form a mixed disulfide via addition of -SCH2CH2X to the reduced sulfhydryl of cysteine, where X is either SO3- or N(CH3)3+ for MTSES and MTSET, respectively (22.Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 23.Akabas M.H. Kaufmann C. Cook T.A. Archdeacon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar, 24.Akabas M.H. Kaufmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 25.Kurz L.L. Zuhlke R.D. Zhang H.-J. Joho R.H. Biophys. J. 1995; 68: 900-905Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 26.Javitch J.A. Fu D. Chen J. Karlin A. Neuron. 1995; 14: 825-831Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 27.Cheung M. Akabas M.H. Biophys. J. 1996; 70: 2688-2695Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Importantly, as reported previously, we (14.Kaplan R.S. Mayor J.A. Brauer D. Kotaria R. Walters D.E. Dean A.M. J. Biol. Chem. 2000; 275: 12009-12016Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and others (28.Holmgren M. Liu Y. Xu Y. Yellen G. Neuropharmacology. 1996" @default.
• W2039717152 created "2016-06-24" @default.
• W2039717152 creator A5007818659 @default.
• W2039717152 creator A5016247362 @default.
• W2039717152 creator A5022281589 @default.
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• W2039717152 creator A5039497533 @default.
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• W2039717152 date "2004-01-01" @default.
• W2039717152 modified "2023-10-14" @default.
• W2039717152 title "The Mitochondrial Citrate Transport Protein" @default.
• W2039717152 cites W1479710013 @default.
• W2039717152 cites W1502473233 @default.
• W2039717152 cites W1531638822 @default.
• W2039717152 cites W1566163765 @default.
• W2039717152 cites W1978294917 @default.
• W2039717152 cites W1988895449 @default.
• W2039717152 cites W1993707260 @default.
• W2039717152 cites W1994373739 @default.
• W2039717152 cites W1994522080 @default.
• W2039717152 cites W2004955668 @default.
• W2039717152 cites W2007431480 @default.
• W2039717152 cites W2009636878 @default.
• W2039717152 cites W2012065308 @default.
• W2039717152 cites W2012622920 @default.
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