Extended Data Fig. 4: In yeast cells, UCP2 catalyses the export of aspartate out of mitochondria in exchange for phosphate.
a, The yeast mitochondrial aspartate/glutamate (AGC1p) and the two isoforms of oxoadipate carrier (ODC1p/ODC2p), which exchange 2-ketoglutarate against malate, are the mitochondrial carriers involved in the yeast malate-aspartate shuttle (MAS) required for the NADH oxidation derived from the peroxisomal oxidation of oleate. b, The UCP2 aspartate/Pi exchange activity together with the glutamate + H+ symport catalysed by the yeast YMC2p restores the loss of AGC1p in MAS. c, The bacterial expressed UCP2R279Q mutant does not catalyse any aspartate/Pi exchange activity once reconstituted into liposomes. Values represent means ± SD of three independent experiments. d, A representative immunoblot of expression of UCP2 and UCP2R279Q V5-tagged proteins in W303 yeast cells used in panel e, similar results were obtained in three biologically independent experiments and in the yeast models reported in Fig. 2i–k. A yeast anti-porin antiserum was used for protein normalization. Porin normalization was carried out on the same SDS-PAGE used to assay V5-tagged UCP2 expression levels (d). e, Serial dilutions of different yeast cell models expressing UCP2 or UCP2R279Q were spotted on YP plate in the presence of oleate. f, To rescue the growth defect of the ΔAGC1 yeast strain on oleate, UCP2 requires the presence of the mitochondrial isoform of the aspartate transaminase (AAT1). g, h, In the inner mitochondrial membrane UCP2 catalyses only the aspartate/Pi exchange reaction. The two models show other two possible exchange activities catalysed by the recombinant UCP2 into liposomes, aspartate/malate and malate/oxaloacetate, which were not confirmed in the yeast models used. Asp, aspartate; Glu, glutamate; Mal, malate; α-KG, α-ketoglutarate; OA, oxaloacetate, MDH, malate dehydrogenase; GDH glutamate dehydrogenase.
