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• W2092573224 abstract "OxlT, the oxalate:formate antiporter ofOxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 → Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate. OxlT, the oxalate:formate antiporter ofOxalobacter formigenes, has a lone charged residue, lysine 355 (Lys-355), at the center of transmembrane helix 11 (TM11). Because Lys-355 is the only charged residue in the hydrophobic sector, we tested the hypothesis that lysine 355 contributes to the binding site for the anionic substrate, oxalate. This idea was supported by mutational analysis, which showed that of five variants studied (Lys-355 → Cys, Gly, Gln, Arg, or Thr), residual function was found for only the K355R derivative, in which catalytic efficiency had fallen 2,600-fold. Further insight came from a study of TM11 single-cysteine mutants, using the impermeant, thiol-specific reagents, carboxyethyl methanethiosulfonate and ethyltrimethylammonium methanethiosulfonate. Of the five reactive positions identified in TM11, four were at the cytoplasmic or periplasmic ends of TM11 (S344C and A345C, and G366C and A370C, respectively), whereas the fifth was at the center of the helix (S359C). Added study with carboxyethyl methanethiosulfonate and ethylsulfonate methylthiosulfonate showed that the attack on S359C could be blocked by the presence of the substrate, oxalate, and that protection could be predicted quantitatively by a kinetic model in which S359C is accessible only in the unliganded form of OxlT. Parallel study showed that the proteoliposomes used in such work contained OxlT of right side-out and inside-out orientations in about equal amounts. Accordingly, full inhibition of S359C by the impermeable methanethiosulfonate-linked probes must reflect an approach from both the cytosolic and periplasmic surfaces of the protein. This, coupled with the finding of substrate protection, leads us to conclude that S359C lies on the translocation pathway through OxlT. Since position 359 and 355 lie on the same helical face, we suggest that Lys-355 also lies on the translocation pathway, consistent with the idea that the essential nature of Lys-355 reflects its role in binding the anionic substrate, oxalate. transmembrane carboxyethyl methanethiosulfonate ethyltrimethylammonium methanethiosulfonate ethylsulfonate methylthiosulfonate 3-(N-maleimidylpropionyl)biocytin octyl-β-d-glucopyranoside 4-(2-aminoethyl)-benzenesulfonyl fluoride phenylmethylsulfonyl fluoride inside-out right side-out 4-morpholinepropanesulfonic acid major facilitator superfamily In the anaerobic bacterium, Oxalobacter formigenes, the proton-motive force is generated by the combined action of an internal oxalate decarboxylation system and the electrogenic oxalate2−/formate1− antiporter, OxlT (1Anantharam V. Allison M.J. Maloney P.C. J. Biol. Chem. 1989; 264: 7244-7250Abstract Full Text PDF PubMed Google Scholar, 2Ruan Z.-S. Anantharam V. Crawford I.T. Ambudkar S.V. Rhee S.Y. Allison M.J. Maloney P.C. J. Biol. Chem. 1992; 267: 10537-10543Abstract Full Text PDF PubMed Google Scholar, 3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 4Maloney P.C. Curr. Opin. Cell Biol. 1994; 6: 571-582Crossref PubMed Scopus (66) Google Scholar). OxlT therefore plays a pivotal role in construction of a “virtual” proton pump, an organizational scheme with relevance to several aspects of microbial cell biology (4Maloney P.C. Curr. Opin. Cell Biol. 1994; 6: 571-582Crossref PubMed Scopus (66) Google Scholar, 5Harold F.M. Maloney P.C. Neidhardt F. Curtis III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 283-306Google Scholar, 6Maloney P.C. Wilson T.H. Neidhardt F. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 1130-1148Google Scholar). Biochemical study (2Ruan Z.-S. Anantharam V. Crawford I.T. Ambudkar S.V. Rhee S.Y. Allison M.J. Maloney P.C. J. Biol. Chem. 1992; 267: 10537-10543Abstract Full Text PDF PubMed Google Scholar, 7Maloney P.C Anantharam V. Allison M.J. J. Biol. Chem. 1992; 267: 10531-10536Abstract Full Text PDF PubMed Google Scholar, 8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) suggests that OxlT, a member of the major facilitator superfamily (MFS) (9Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar), may also serve as a valuable model for more broadly directed studies of membrane transport proteins. For such reasons, further study of OxlT may contribute to ongoing studies of both the biology and biochemistry of membrane transport. Hydropathy analysis of the OxlT amino acid sequence (3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), together with circular dichroism spectroscopy of the purified, solubilized protein (8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), suggests the presence of 12 transmembrane α-helices, a characteristic shared by most other members of the major facilitator superfamily (9Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar). This analysis also predicts that Lys-355 is positioned near the center of transmembrane helix 11 (TM11)1 (3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), an expectation verified by site-directed fluorescent labeling of single-cysteine variants in this vicinity (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Direct evaluation of OxlT topology 2Ye, L., Jia, Z., Jung, T., and Maloney, P. C. (2001) J. Bacteriol., in press. suggests that Lys-355 does not interact with a nearby anionic residue, thereby raising an apparent paradox. Placement of an uncompensated charge within a hydrophobic environment adds a destabilizing element to membrane protein structure (12Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar, 13Engelman D.M. Steitz T.A. Goldman A. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 321-353Crossref PubMed Scopus (1201) Google Scholar), but OxlT can be remarkably stable in lipid-detergent micelles (7Maloney P.C Anantharam V. Allison M.J. J. Biol. Chem. 1992; 267: 10531-10536Abstract Full Text PDF PubMed Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This contradiction might be reconciled if the energetic disadvantage contributed by Lys-355 were used to offset the energetic cost of binding and/or transporting substrate anions. Such compensating effects could be achieved in a simple way if TM11 forms part of the substrate translocation pathway, allowing direct interactions between positively charged Lys-355 and negatively charged substrate. That solubilized OxlT is intensely stabilized by the presence of its substrate anions (7Maloney P.C Anantharam V. Allison M.J. J. Biol. Chem. 1992; 267: 10531-10536Abstract Full Text PDF PubMed Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), gives indirect support to this view, but more concrete observations are necessary if this model is to guide further work. The experiments reported here sought to address such issues by asking if one or more residues on TM11 is involved in substrate binding and/or translocation. To do this, we first examined the role of Lys-355 itself by analysis of site-specific mutants. We then exploited a panel of single-cysteine variants to search throughout TM11 for perturbations attributable to either cysteine substitution or to site-specific chemical modification. Together, these two lines of study provide evidence justifying the idea that as substrate passes through OxlT, it encounters residue(s) along TM11 in the vicinity of Lys-355. Wild type OxlT was encoded within a 1.4-kilobase XbaI-HindIII fragment in pBluescript II SK+; expression of OxlT was regulated by thelac promoter (3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This plasmid also served as the vehicle for site-directed mutagenesis using a double-stranded protocol (Cameleon™, Stratagene) to generate mutants of Lys-355. Similar techniques were used to construct a fully active cysteine-less variant with nine tandem histidine residues added at its C terminus to facilitate protein purification (8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This latter derivative was used as the template for generation of the panel of single-cysteine derivatives spanning TM11 (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). OxlT and its mutants were carried inEscherichia coli strain XL-1, together with plasmid pMS421 (Specr, LacIq) to limit inappropriate basal expression (3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). OxlT expression was induced by addition of 0.5 mm isopropyl-1-thio-β-d-galactopyranoside to cells in the mid-exponential phase of growth; cells were harvested after an additional 4 h growth. Membrane ghosts, prepared as described (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 14Varadhachary A. Maloney P.C. Mol. Microbiol. 1990; 4: 1407-1411Crossref PubMed Scopus (18) Google Scholar), were suspended in distilled water; membrane proteins were solubilized by incubating for 20 min on ice in Buffer A (20 mm MOPS/K (pH 7), 20% (v/v) glycerol, 4.2 mg/ml E. coli phospholipid, 10 mm potassium oxalate, and 1.5% (w/v) octylglucoside). Here, and in all work involving thiol labeling (below), the E. coli lipid used for solubilization and reconstitution had been hydrated with distilled water rather than with the usual 2 mm β-mercaptoethanol (1Anantharam V. Allison M.J. Maloney P.C. J. Biol. Chem. 1989; 264: 7244-7250Abstract Full Text PDF PubMed Google Scholar). Solubilized protein (0.7–1 mg/ml) was reconstituted by detergent dilution (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Ambudkar S.V. Maloney P.C. J. Biol. Chem. 1986; 261: 10079-10086Abstract Full Text PDF PubMed Google Scholar) to give proteoliposomes loaded with 100 mm potassium oxalate, 50 mm MOPS/K (pH 7). In most cases, [14C]oxalate transport was measured by a rapid filtration assay. Triplicate 100–200-μl aliquots of proteoliposomes were placed at the center of Millipore GSTF filters (0.22-μm pore size), washed twice with 5 ml of Buffer B (100 mm potassium sulfate, 50 mm MOPS/K, (pH 7) to remove external (unlabeled) oxalate, and after interrupting the vacuum, proteoliposomes trapped on the filter were overlaid with 300 μl of Buffer B containing 100 μm [14C]oxalate. Unless otherwise indicated, the assay was terminated after 20 s by filtration, followed with two washes using 5 ml of iced Buffer B. Depending on experimental design, oxalate transport is given as a fraction of the maximum incorporation or as relative to that of the cysteine-less parental protein, after correction for differences in protein expression (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This assay provides a wider dynamic range than available earlier (3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and is especially suited to the parallel study of a large number of samples. [14C]Oxalate transport was also monitored by a traditional filtration assay (1Anantharam V. Allison M.J. Maloney P.C. J. Biol. Chem. 1989; 264: 7244-7250Abstract Full Text PDF PubMed Google Scholar, 3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In these cases, washed proteoliposomes suspended as a concentrated stock in Buffer B were diluted 10-fold into this same buffer at 23 °C, and the transport reaction was initiated by addition of either 100 μm or 1 mm labeled substrate, as specified. At timed intervals, samples were withdrawn for filtration and washing. To screen for thiol reactivity in the TM11 panel of single-cysteine derivatives, membrane ghosts were placed with excess probe (2 mm MTSCE or 1 mm MTSET) in 20 mm potassium phosphate (pH 8) for 1 h at 23 °C. Unreacted probe was removed either by a quench using 5 mmβ-mercaptoethanol, or by four cycles of washing with 20 mm potassium phosphate (pH 7), as noted. Subsequently, membrane protein was solubilized and reconstituted as described above. These conditions allow labeling of residues on both intra- and extracellular surfaces2 (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The orientation of OxlT in proteoliposomes was deduced from the pattern of thiol labeling of cysteines placed at either end of TM11 and also by monitoring the effects on substrate transport of external trypsin. For thiol labeling, the target protein (A345C or A370C) was purified (>95%) by Ni2+-nitrilotriacetic acid affinity chromatography, essentially as described (8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) except that the final elution used a buffer containing 100 mm potassium oxalate, 50 mm potassium acetate, 20% glycerol (v/v), and 5 mg/ml diheptanoylphosphatidylcholine (pH 4.5). Prior to reconstitution, 10 μl of the eluate (5–10 μg of protein), was mixed with 240 μl of Buffer A in which lipid was increased to 10 mg/ml to compensate for that normally contributed by native membranes (see above). Protein concentration (40 μg/ml) was close to that of OxlT in crude extracts, which contained the transporter at 3–5% of membrane protein (8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). After reconstitution, proteoliposomes were washed and finally suspended in 0.5 ml of Buffer B. Cysteines exposed to the suspending medium were blocked by exposure to 2 mm MTSET for 15 min at 23 °C. The probe was removed by a 40-fold dilution with iced Buffer B, followed by centrifugation and resuspension of proteoliposomes to their original volume in Buffer B. MTSET-pretreated proteoliposomes, along with untreated controls, were then incubated with either 400 μm MPB or an equivalent volume of its dimethyl sulfoxide solvent for 60 min at 23 °C. This second reaction was quenched by dilution, centrifugation, and washing, as above. In some cases, MPB labeling was followed by addition of trypsin (1 mg/ml) and further incubation for 90 min at 33 °C, after which protease action was quenched by dilution, centrifugation, and resuspension (as above) using iced Buffer B containing 1 mm AEBSF. After SDS-polyacrylamide gel electrophoresis and after transfer to nitrocellulose, the presence of MPB was detected by enhanced chemiluminescence (Pierce, SuperSignal™) using a horseradish peroxidase-linked anti-biotin antibody (New England Biolabs). To monitor recovery of OxlT, the blot was stripped and reprobed with a polyclonal antibody reactive to the OxlT N terminus (2Ruan Z.-S. Anantharam V. Crawford I.T. Ambudkar S.V. Rhee S.Y. Allison M.J. Maloney P.C. J. Biol. Chem. 1992; 267: 10537-10543Abstract Full Text PDF PubMed Google Scholar, 3Abe K. Ruan Z.-S. Maloney P.C. J. Biol. Chem. 1996; 271: 6789-6793Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Signals were captured by an automated documentation system (Fuji Medical Systems, Inc.). The sensitivity of OxlT to external trypsin was monitored using the wild-type protein purified as described (8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). After reconstitution (see above), proteoliposomes were placed in 1 ml of Buffer B with and without trypsin (1 mg/ml) at 33 °C, as described (16Fann M.-C. Maloney P.C. J. Biol. Chem. 1998; 273: 33735-33740Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). At timed intervals, samples were diluted 25-fold with iced Buffer B containing 0.5 mm PMSF. After centrifugation and resuspension, as above, initial rates of [14C]oxalate transport were determined using the traditional filtration assay with 100 μm labeled substrate. Membrane ghosts prepared using the S359C mutant were extracted and crude extracts were reconstituted as noted above. To characterize the concentration dependence of inhibition by MTS-linked probes, proteoliposomes trapped on a Millipore GSTF filter (0.22-μm pore size) were washed twice with Buffer B and then overlaid (in triplicate) for 5 min with 0.5 ml of Buffer B containing MTSCE, MTSES, or MTSET at the specified concentrations. When protection by substrate was monitored, the 5-min incubation was in the presence of probe and increasing concentrations of potassium oxalate (from 0 to 100 mm); a reciprocal decrease in potassium sulfate (from 100 to 0 mm) served to maintain constant ionic and osmotic strength. Reactions were terminated by rapid filtration, followed by two washes with Buffer B, and residual OxlT function was monitored by a second overlay with labeled substrate, as noted above. Preliminary trials showed that half-maximal inhibition of OxlT required 10–30 μm MTSCE, and that nearly complete inhibition (≥95%) was achieved by raising probe concentration to 300 μm. However, even at such high levels of MTSCE, sufficiently high concentrations of oxalate afforded complete protection. Because 2 mm MTSCE had no effect on oxalate transport by the cysteine-less parent (not shown), inhibition of the S359C derivative was attributed solely to reaction of the probe with the target cysteine at position 359, justifying use of a simple kinetic scheme to model probe inhibition. We assumed unliganded OxlT (C) could interact with either substrate (S) or the probe (P), generating, respectively, either liganded OxlT (CS) or an irreversibly inhibited complex (CP*). C+S↔CSC+P→CP*REACTIONS1AND2 Because inhibition by MTSCE occurs on a time scale of minutes, while turnover for OxlT-mediated exchange takes place in milliseconds or less (1Anantharam V. Allison M.J. Maloney P.C. J. Biol. Chem. 1989; 264: 7244-7250Abstract Full Text PDF PubMed Google Scholar, 2Ruan Z.-S. Anantharam V. Crawford I.T. Ambudkar S.V. Rhee S.Y. Allison M.J. Maloney P.C. J. Biol. Chem. 1992; 267: 10537-10543Abstract Full Text PDF PubMed Google Scholar, 8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we also assumed rapid equilibrium between OxlT and its substrate, as described in earlier kinetic schemes (7Maloney P.C Anantharam V. Allison M.J. J. Biol. Chem. 1992; 267: 10531-10536Abstract Full Text PDF PubMed Google Scholar, 8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). With these assumptions, the model makes the following quantitative prediction concerning the parameter F, the fraction of the OxlT population that remains unmodified by MTSCE.−lnF=kPt/(1+S/KD)Equation 1 or−1/lnF=(1/kPt)+(1/KD)(1/kPt)SEquation 2 S and P represent substrate (oxalate) and probe concentrations, respectively, t is time, k is the rate constant governing probe modification (Equation 2), andKd is the dissociation constant for the liganded complex, CS. This model was used to evaluate probe interactions at Ser-359, using material purified as described above for the A345C and A370C proteins. Purified E. coli phospholipid was obtained as a lyophilized powder from Avanti Polar Lipids, Inc. MTSCE, MTSET, and MTSES were from Toronto Research Chemicals, Inc., and MPB was from Molecular Probes, Inc. Roche-Calbiochem provided octylglucoside, while the trypsin (tosylphenylalanyl chloromethyl ketone-treated) was from Worthington. PMSF and AEBSF were from Sigma. [14C]Oxalate was from PerkinElmer Life Sciences. Two classes of experiments explored the idea that Lys-355 and other residues on TM11 might be associated with substrate binding and translocation. In one group of studies, described immediately below, the role of Lys-355 itself was examined by noting the behavior of its mutants. In a second group of experiments, cysteine-scanning mutagenesis identified a residue, near to Lys-355, which served as an informative target for cysteine-directed agents. Both lines of study provided evidence consistent with the idea that Lys-355 and residues in its vicinity normally serve to aid in the translocation of OxlT substrates. If Lys-355 is essential to OxlT function, substitutions at this position should compromise activity. To test this prediction, we used site-directed mutagenesis to replace Lys-355 with alternate residues, including cysteine, glycine, glutamine, arginine, and threonine. In the usual assays of transport by OxlT (see “Experimental Procedures”), these mutants displayed little or no function. In one case (K355G) this was attributable to lack of expression; all other mutants were found in membranes in amounts comparable to the wild type (data not shown; see Ref. 10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), so that lack of function was due to failure of OxlT itself. We considered the possibility that such null responses might reflect a poor substrate affinity, and for that reason we also measured transport by oxalate-loaded proteoliposomes using external [14C]oxalate at 1 mm rather than the usual 0.1 mm. In only one case (the K355R variant) did this added test reveal a significant activity (Fig.1), although the time course of transport by the mutant was greatly extended relative to that of the parental wild type protein. In separate experiments, we performed a further, kinetic analysis of the K355R derivative. That work showed this mutant to have both an elevated Michaelis constant (Km of 5.4 versus 0.15 mm) and a reduced maximal velocity (Vmax of 66 versus 4,700 nmol/min/mg protein), leading to a 2,600-fold reduction in catalytic efficiency (Vmax/Km) relative to the parental protein. Accordingly, because mutants of Lys-355 retained little or no function, we conclude this residue is essential for normal OxlT function. The behavior of Lys-355 mutants (Fig. 1) suggests that OxlT requires (at least) the presence of positive charge at position 355. To ask whether nearby residues might also have functional significance, we analyzed a panel of TM11 single-cysteine variants that had earlier been used to establish topological relationships in this region (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We first tested [14C]oxalate transport under initial rate conditions during the oxalate self-exchange reaction (Fig.2 A). The N347C, A368C, and I369C variants were too poorly expressed (≤5% the parental level) for this analysis, but all others were present at levels high enough (≥40% normal) (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) to assess functional status. Among the 27 expressed proteins, two (G349C and K355C) gave undetectable levels of [14C]oxalate transport; three others (A354C, G362C, G363C) gave marginal responses (0.2–1% residual activity). In all other cases, we found activity corresponding to at least 1% of the cysteine-less parental protein, yielding a signal-to-noise ratio of at least 20. Since the turnover number for OxlT is at least 1000-fold greater than usually observed for membrane transporters (1Anantharam V. Allison M.J. Maloney P.C. J. Biol. Chem. 1989; 264: 7244-7250Abstract Full Text PDF PubMed Google Scholar, 4Maloney P.C. Curr. Opin. Cell Biol. 1994; 6: 571-582Crossref PubMed Scopus (66) Google Scholar, 8Fu D. Maloney P.C. J. Biol. Chem. 1997; 272: 2129-2135Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), even these low relative levels of activity might reflect significant rates of anion exchange. Such findings, including the null behavior of K355C, are consistent with earlier studies (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and with the results noted above (Fig. 1). In earlier work with this same set of single-cysteine variants, we showed that the TM11 core region (positions 351–361; Fig.2 A) is inaccessible to Oregon Green maleimide, a hydrophilic thiol-reactive agent of moderate size (∼500 daltons) (10Fu D. Maloney P.C. J. Biol. Chem. 1998; 273: 17962-17967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The present work shows that, although cysteine substitutions giving reduced function (≤10% parental) are broadly represented throughout TM11, a striking distribution is found in this inaccessible region. Within this core, cysteine substitutions yielding low specific activity are restricted to the helical face containing Lys-355 (Fig. 2 B), indicating a distinct functional asymmetry in this area. The high velocity of OxlT-mediated reactions ensures that even cysteine substitution mutants with low residual activity (Fig. 2 A) show satisfactory signal-to-noise ratios. For this reason we pursued a study of these variants using the thiol-specific probes, MTSCE and MTSET. These agents were chosen for their specificity in modification of cysteine (17Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (545) Google Scholar), because their polar and linear character increases the probability they might have access to regions within OxlT near a substrate binding region (e.g. possibly near Lys-355), and because they generate reaction products of a clearly distinct character-MTSET implants a fixed positive charge (RS-SCH2CH2N(CH3)4+), whereas the reaction with MTSCE results in appearance of a carboxyl group that would carry negative charge at pH 7 (RS-SCH2CH2COO−). Membranes containing TM11 single-cysteine variants were exposed to excess MTSCE or MTSET, with and without tests for reversibility by later exposure to β-mercaptoethanol. Subsequent to this in situ labeling, protein was solubilized, and assays of transport by proteoliposomes recorded residual function. MTSET and/or MTSCE gave significant inhibition at five positions, two each in the TM11 cytoplasmic (S344C, A345C) and perip" @default.
• W2092573224 created "2016-06-24" @default.
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• W2092573224 creator A5041603149 @default.
• W2092573224 creator A5049014516 @default.
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• W2092573224 date "2001-03-01" @default.
• W2092573224 modified "2023-09-29" @default.
• W2092573224 title "Structure/Function Relationships in OxlT, the Oxalate-Formate Transporter of Oxalobacter formigenes" @default.
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