Oligomeric state of the oxalate transporter, OxlT

Abstract

OxlT, the oxalate transporter of Oxalobacter formigenes, was studied to determine its oligomeric state in solution and in the membrane. Three independent approaches were used. First, we used triple-detector (SEC-LS) size exclusion chromatography to analyze purified OxlT in detergent/lipid micelles. These measurements evaluate protein mass in a manner independent of contributions from detergent and lipid; such work shows an average OxlT mass near 47 kDa for detergent-solubilized material, consistent with that expected for monomeric OxlT (46 kDa). A disulfide-linked OxlT mutant was used to verify that it was possible detect dimers under these conditions. A second approach used amino-reactive cross-linkers of varying spacer lengths to study OxlT in detergent/lipid micelles and in natural or artificial membranes, followed by analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These tests, performed under conditions where the presence of dimers can be documented for either of two known dimeric transporters (AdiC or TetL), indicate that OxlT exists as a monomer in the membrane and retains this status upon detergent solubilization. In a final test, we showed that reconstitution of OxlT into lipid vesicles at variable protein/lipid ratios has no effect on the specific activity of subsequent oxalate transport, as the OxlT content varies between 0.027 and 5.4 OxlT monomers/proteoliposome. We conclude that OxlT is a functional monomer in the membrane and in detergent/lipid micelles.

Figure 1. OxlT molecular weight determination using TD-SEC-LS

(A) 50-100 μg of OxlT was loaded onto a Shodex kw-803 HPLC column in the presence of 5% glycerol, 50 mM potassium MOPS, 100 mM potassium oxalate and 0.1% DDM (pH 7). The tracings show normalized outputs of detectors for measurement of A₂₈₀ (UV, black), Light Scattering (LS, red) and Refractive Index (RI, blue). In a parallel trial in the same experiment and using the same buffer conditions, the instrument calibration constant K₁ was obtained using a reference set of six protein standards of mass between 29 kDa and 141 kDa (see Experimental Procedures). (B) For illustrative purposes, using data from Part A, OxlT mass (red trace) was calculated for the region across the A₂₈₀ trace at ca. 75% peak height. For this and other experiments, the mass values reported in Tables 1 and 2 were calculated at the peak of the A₂₈₀ trace.

Figure 2. Cross-linking of a cysteine substitution mutant

The OxlT H413C variant was subjected to TD-SEC-LS or SEC after solubilization and purification. (A) Two forms of OxlT were detected by TD-SEC-LS in 0.1% DDM at pH 7. Based on TD-SEC-LS measurement, the calculated molecular masses of these forms are 103 kDa and 56 kDa, corresponding approximately to dimer or monomer forms of OxlT. (B) The dimer form was not found after addition of reducing agent (10 mM TCEP). In both parts A and B, parallel chromatographic runs without protein were used to identify the elution time of the DDM micelle, as reflected by its RI trace (dotted lines). (C) SEC using Superdex 200 was performed after overnight incubation at 23°C at pH 8.5, so as to facilitate disulfide bond formation. A sample of 100 μg protein was loaded onto the column in elution buffer (pH 7.5) containing 5% glycerol, 20 mM Tris/HCl, 100 mM NaCl, 100 mM potassium oxalate, and 0.1% DDM, with (dotted trace) or without (solid trace) 10 mM TCEP. (D) Non-reducing SDS-PAGE of samples taken from the apparent dimer and monomer peaks showed the presence of OxlT species corresponding to ca. 70 kDa and 35 kDa, respectively.

Figure 3. Amino-directed cross-linking of solubilized OxlT

After purification, AdiC and OxlT were each suspended (50 μg/ml) in buffer (pH 7.5) with 100 mM potassium oxalate, 20 mM potassium phosphate, 20% (w/v) glycerol, 0.1% DDM, and exposed to the indicated cross-linking agent for 30 min at 23°C. The reaction was quenched by addition of Tris/Cl (pH 7.5) to 50 mM, and samples were processed for SDS-PAGE. Protein was detected using Silver Stain Plus (BioRad); standard markers on the same gels were used to extrapolate the mass values indicated. (A) Samples were exposed to 2 mM DSG, DSP or EGS. (B) Samples were exposed to increasing concentrations of EGS (0 to 2 mM), in the absence or presence of 0.5 % SDS, as indicated. (C) Samples were collected before and after addition of DSG, DSP or EGS, as in Part A, to evaluate residual function by reconstitution. The observed initial rates of [¹⁴C]oxalate transport are reported relative to that found for the untreated sample. (D) Samples were treated with 100 mM glutaraldehyde.

Figure 4. Amino-directed cross-linking of membrane-embedded OxlT

(A) Washed membranes from cells expressing either AdiC or OxlT were suspended in buffer (pH 7.5) containing 100 mM potassium oxalate, 50 mM potassium phosphate and then treated with 0-10 mM EGS for 30 min at 23C. After the reaction was quenched, samples were taken for SDS-PAGE (see legend to Fig. 3). (B) In a parallel experiment using the same conditions, samples were taken for reconstitution to evaluate residual OxlT function, as described in the legend to Figure 3C. (C) Purified OxlT or AdiC was reconstituted into proteoliposomes prepared using at 50/50 mixture of palmitoyloleoylphosphatidyl choline and palmitoyloleoylphosphatidyl glycerol. Cross-linking was assessed by treatment with 100 mM glutaraldehyde, using AdiC as the positive control.

Figure 5. Reconstitution of OxlT at low protein-to-lipid ratios

Initial rates of [¹⁴C]oxalate transport were measured after reconstitution of OxlT at variable protein-to-lipid ratios. Each reconstitution mixture contained 2.7 mg phospholipid together with 0.01 to 2 μg OxlT, achieving a nominal monomer-to-proteoliposome ratio between about 0.03 and 6. The mean values of duplicate measurements are given, normalized to the value obtained at the highest dose of OxlT. The line drawn reflects the theoretical expectation if OxlT specific activity is constant with OxlT content.