Supplementary Figure 2: Functional properties.

Gel filtration and light scattering results for different protein constructs purified in the detergent DM. Continuous black traces correspond to the UV₂₈₀ elution profiles. The respective molecular weight of the protein component obtained from light scattering is shown at its corresponding position on the chromatogram in red. Panels show (a), SLC26Dg, and (b), the SLC26Dg-Nb5776 complex. (c) Glutaraldehyde (GA) crosslinking of reconstituted SLC26Dg. Samples were separated by SDS PAGE, the respective molecular weight of marker proteins is indicated. The band at around 130 kDa corresponds to a cross-linked SLC26Dg dimer, the band at around 55 kDa to monomeric SLC26Dg observed upon solubilization of proteoliposomes in SDS prior to glutaraldehyde addition. Left panel, SDS-PAGE gel of proteoliposomes reconstituted at a protein:lipid ratio of 1:50 (wt/wt); right panel, Western blot of proteoliposomes reconstituted at 1:250 and 1:500 indicating that crosslinking of SLC26Dg dimers also occurred at very low protein concentrations in the membrane. (d) Substrate selectivity. Competition of ¹⁴C-fumarate uptake by a 70-fold excess of other unlabeled dicarboxylates. (e) Concentration dependence of fumarate transport. The solid curve shows a fit to a Michaelis-Menten equation with a linear component due to the passive uptake of fumarate into liposomes. The dashed line shows the slope of the linear component. Specific activities were calculated assuming a complete incorporation of the protein into liposomes during reconstitution. (f) Membrane orientation of SLC26Dg (WT) and SLC26Dg^ΔSTAS (TM) in proteoliposomes. Both proteins were expressed and reconstituted as N-terminal fusions to GFP. The membrane orientation was investigated by probing the accessibility of the HRV 3C protease cleavage site between the membrane transporter and GFP. In-gel fluorescence was measured after separation of the cleaved products on SDS PAGE. – refers to the untreated samples, p to samples treated with HRV 3C protease, and p/d to samples treated with HRV 3C protease and detergent. * indicates uncleaved protein. For both constructs, incubation with the protease decreased the GFP fluorescence of the uncleaved protein by approximately 50% indicating a similar random distribution of both orientations within the membrane. Molecular weight of markers (kDa) are indicated on the left. (g) Transport properties of SLC26Dg^ΔSTAS. Time dependent uptake of ¹⁴C-fumarate into proteoliposomes containing SLC26Dg^ΔSTAS (TM). ΔpH refers to an outside pH of 6.0 and a pH of 7.5 inside the liposomes, ΔNa⁺ to an external Na⁺ concentration of 50 mM and no Na⁺ inside the liposomes. Traces of WT and liposomes not containing any protein (with data presented in Fig. 1a) are shown for comparison. Proteoliposomes were prepared with equimolar amounts of protein. Equimolar protein amounts were also used for each data point. Data in d and g represents mean and s.e.m of 3 technical replicates, data in e represent mean and s.e.m of 6 technical replicates from two independent batches of proteoliposomes.