(A) Structure of the GltPh trimer (pdb: 2nwx) is shown in ribbon representation. (B) Flow chart outlining the strategy used for the dissociation and reassociation of the GltPh trimer. (C) Size exclusion chromatography of the native GltPh (black) and the SDS dissociated GltPh following dilution into lipid vesicles (red) and into DDM (blue). Reassembly of GltPh takes place in lipid vesicles. Inset, SDS-PAGE gel showing the glutaraldehyde crosslinking of the native GltPh (lanes 1–3), reassociated GltPh (r-GltPh, lanes 4–6) and the SDS dissociated GltPh subunits (lanes 7–9). Lanes 1, 4, and 7, without glutaraldehyde crosslinking, lanes 2, 5, and 8, with glutaraldehyde crosslinking and lanes 3, 6, and 9, with glutaraldehyde crosslinking in the presence of 1% SDS. The oligomeric nature of the protein band (1x, 2x, and 3x) is indicated. (D) Asp binding by native and r-GltPh. Asp binding was monitored by changes in fluorescence of the native (black circles) or r-GltPh (red squares) with the L130W substitution. The fraction of the protein bound (Fbound) is determined by dividing the change in fluorescence upon addition of Asp by the total change at the end of the titration. Solid lines are fits to the data using the equation described in Materials and methods with KD values of 183 ± 16 nM for native GltPh and 216 ± 15 nM for r-GltPh. The binding assays were conducted in 10 mM Na+. (E) Asp uptake assay. The time course of [14C]-Asp uptake for native GltPh (black circles) and r-GltPh (red squares) in the presence of a Na+ gradient. No uptake is observed for native GltPh (black diamonds) and r-GltPh (red triangles) in the absence of a Na+ gradient. For panels D and E, error bars indicate standard error of mean (SEM) for n > 3.