Figure 3: The p.(Lys232Arg) mutation interferes with the formation of the matrix salt bridge network and impairs function of the mitochondrial oxodicarboxylate carrier (SLC25A21).
(a) Membrane view of the comparative homology model of human SLC25A21 generated with SwissModel (Arnold et al.,41) based on the structure of the bovine adenosine diphosphate/adenosine triphosphate carrier (alignment shown in Supplementary File S1, Supplementary Figure S1). The matrix and cytoplasmic salt bridge network are shown in dark blue and cyan, respectively. The mutated lysine 232 is shown in yellow. The residues of the proposed substrate binding site are shown in green and the substrate 2-oxoglutarate is shown in an orange ball and stick representation. (b) Cytoplasmic view showing the residues involved in the formation of the matrix salt bridge network in dark blue. The most probable side-chain conformations of the p.(Lys232Arg) mutation are shown in magenta, indicating that the substitution might impair binding to Glu131 and might lead to other polar interactions. (c) The 2-oxoglutarate uptake curves of SLC25A21 and SLC25A21_p.(Lys232Arg). Fused membrane vesicles of Lactococcus lactis expressing SLC25A21 (triangles) or SLC25A21_p.(Lys232Arg) (circles) were preloaded with 5 mM 2-oxoglutarate and transport was initiated with the external addition of 1.5 μM [14C]-2-oxoglutarate. Membranes of the uninduced SLC25A21 strain (squares) show background binding, indicating that SLC25A21_p.(Lys232Arg) does not transport above background. (d) Expression levels of the wild-type and mutant SLC25A21 were similar, indicating the mutation did not affect expression and insertion of the carrier in lactococcal membranes. (e) The 2-oxoglutarate transport rates of SLC25A21 and SLC25A21_p.(Lys232Arg) after correction for background binding. The error bars represent the standard deviation of four technical repeats. Student t-tests, significant uptake above background: P > 0.05, not significant (NS); *P < 0.01; **P < 0.001; ***P < 0.0001; ****P < 0.0001.
