Fig. 1. Bacterial overexpression and purification of citrin, aralar1 and their C-terminal domains. Proteins were separated by SDS–PAGE and either stained with Coomassie Blue dye (A and C) or transferred to nitrocellulose membrane and detected with specific polyclonal antibodies (B, D and E). (A and B) Cells of E.coli CO214(DE3) overexpressing citrin. Samples were taken at induction of expression (lane 1) and 12 h later (lane 2). The same number of bacteria was analyzed in each sample. Lanes 3, 6 µg (A) and 1 µg (B) of purified citrin obtained from bacteria in lane 2 of (A). (C) A 5 µg aliquot of purified citrin (lane 1), its C-terminal domain (lane 2), aralar1 (lane 3) and its C-terminal domain (lane 4). (D) Immunoreaction of 0.2 µg of purified citrin and its C-terminal domain (lanes 1 and 2, respectively) with antiserum to the C-terminal domain. (E) Immunoreaction of 0.2 µg of purified aralar1 and its C-terminal domain (lanes 1 and 2, respectively) with antiserum against the C-terminal domain.

Fig. 2. Lineweaver–Burk plots of the [¹⁴C]aspartate/aspartate and [¹⁴C]glutamate/aspartate exchanges in proteoliposomes reconstituted with citrin. Radioactive aspartate (squares) or glutamate (circles) was added at the concentrations indicated to proteoliposomes containing 20 mM aspartate. Valinomycin (1 µg/mg of phospholipids) and nigericin (50 ng/mg of phospholipids) in ethanol (10 µl/ml of liposomes) (B) or ethanol alone (A) were present in the reaction mixture. All data were determined in one experiment with the same preparation of proteoliposomes. Similar results were obtained in three additional independent experiments.

Fig. 3. Reduction of MTT in cells overexpressing aralar1 or citrin. Digitonin (20 µM)-permeabilized HEK-293T cells overexpressing aralar1 (filled bars) or citrin (striped bars) or the empty pIRES vector (open bars) were tested for reduction of MTT in the presence of either 1 mM glutamate, 5 mM malate and 10 mM lactate (G + M + L) or 15 mM glycerol-3-phosphate and 10 mM lactate (G-P + L). Under basal conditions, no substrates were added. (A) Reduction of MTT in the presence of various substrates. (B and C) Normalized MTT reduction. The results (means ± SEM) correspond to a representative experiment performed four times. The differences between controls and cells overexpressing aralar1 or citrin were significant (*P <0.05, ***P <0.001, one-way ANOVA and Bonferroni t-test).

Fig. 4. The N-terminal domains of aralar1 and citrin containing the Ca²⁺-binding sites are exposed to the cytoplasmic surface of the inner mitochondrial membrane. (A) Schematic representation of the topology of aralar1 or citrin in the inner mitochondrial membrane. In the N-terminal domain, the four EF-hand Ca²⁺-binding motifs are indicated. (B) Proteolysis of aralar1 and citrin with proteinase K. Mitoplasts (1) and mitochondria (2) isolated from rat brain (aralar1) or liver (citrin) were treated with proteinase K as indicated. Each fraction (20 µg protein/lane) was subjected to reaction with an antibody against the N-terminal domain of aralar1 or citrin. As control, analyses were also performed for the β-subunit of F₁-ATPase.

Fig. 5. Decarboxylation of l-[1-¹⁴C]glutamate and [1-¹⁴C]α-ketoglutarate in HEK-293T cells. (A) Pathways for glutamate uptake and decarboxylation in mitochondria. AGC, aspartate/glutamate carrier; OMC, α-ketoglutarate/malate carrier; AOAA, aminooxyacetic acid; GC, glutamate/OH^-- carrier; CaU, Ca²⁺ uniporter; RR, ruthenium red; α-KGDH, α-ketoglutarate dehydrogenase; GDH, glutamate dehydrogenase; LDH, lactate dehydrogenase; mAST, cAST, mMDH and cMDH, mitochondrial and cytosolic aspartate aminotransferases and malate dehydrogenases, respectively. (B) Effects of malate and lactate on the pathway of glutamate decarboxylation. HEK-293T cells (∼10 µg protein/well) were incubated in the presence of 1 mM l-[1-¹⁴C]glutamate (circles), and 5 mM malate (squares) or 5 mM malate + 10 mM lactate (triangles) for the times shown. AOAA (5 mM) was added where indicated (filled symbols). The data correspond to a single experiment performed in triplicate. It has been repeated 2–3 times with similar results. (C) Effects of Ca²⁺ and RR on glutamate decarboxylation in permeabilized cells. HEK-293T cells were incubated with 1 mM l-[1-¹⁴C]glutamate, 5 mM malate, 10 mM lactate and 20 µM digitonin for 60 min in the presence (AOAA-resistant CO₂ production) or absence (total CO₂) of 5 mM AOAA with 20 µM CaCl₂ (filled bars) or 20 µM CaCl₂ + 1 nmol RR/mg protein (striped bars), or without CaCl₂ (open bars). AOAA-sensitive CO₂ production was the difference between total and AOAA-resistant CO₂ production. Results are means ± SEM of five paired experiments performed in triplicate. The difference between incubations with or without Ca²⁺ and incubations with Ca²⁺ with or without RR was significant, where indicated (*P <0.05, Wilcoxon signed rank t-test). (D) Inhibition by RR of Ca²⁺-stimulated α-ketoglutarate decarboxylation in digitonin-permeabilized cells. HEK-293T cells were incubated with 50 µM [1-¹⁴C]α-ketoglutarate, 5 mM malate and 20 µM digitonin for 60 min in the presence of 20 µM CaCl₂ alone, or together with 1 or 2 nmol RR/mg protein, or in the absence of Ca²⁺. Data are means ± SEM of four experiments performed in triplicate. The significance of the difference between incubations with Ca²⁺ and either no Ca²⁺ or Ca²⁺ and RR is indicated (***P <0.001, one-way ANOVA followed by Bonferroni t-test).

Fig. 6. Ca²⁺-induced stimulation of glutamate decarboxylation in HEK-293T cells overexpressing aralar1 or citrin. Cultures of HEK-293T cells expressing aralar1 (filled bars), citrin (striped bars) or empty pIRES vector (open bars) were incubated with 1 mM l-[1-¹⁴C]glutamate, 5 mM malate, 10 mM lactate and 20 µM digitonin for 60 min in the presence (AOAA-resistant ¹⁴CO₂ production) or absence (total ¹⁴CO₂) of 5 mM AOAA, and either 20 µM CaCl₂, 20 µM CaCl₂ + 1 nmol RR/mg protein, or in the absence of Ca²⁺. AOAA-sensitive ¹⁴CO₂ production was the difference between total and AOAA-resistant ¹⁴CO₂ production. The results, expressed as percentage of ¹⁴CO₂ formation in the absence of Ca²⁺, are means ± SEM of 4–9 experiments performed in triplicate. The significance of the difference between cells expressing aralar1 or citrin and controls is indicated (*P <0.05, **P <0.025, Mann–Whitney t-test, ***P< 0.001, one-way ANOVA followed by Bonferroni t-test).