Back to article: Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells

FIGURE 3: Partially decreasing cytosolic translation preserves oxidative phosphorylation in taz1Δ yeast. The experiments here described were performed using mitochondria isolated from cells grown for 24 hours at 36°C in CSM containing 0.5% galactose + 2% ethanol, supple-mented or not as indicated with 10 nM CHX, until a density of 2-3 OD600nm. (A, B) Steady-state levels of proteins involved in the transfer of electrons to oxygen. (A) Proteins were ex-tracted from the mitochondrial samples using 2 g digitonin per g of proteins. The supercomplexes III2-IV2 and III2-IV1 were revealed by the complex IV activity after separation by CN-PAGE or by western blot with antibodies against Cox2 in BN-PAGE gels. (B) Left panel. Total mitochondrial protein samples were resolved by SDS-PAGE (50 μg per lane) and probed with antibodies against the in-dicated proteins. The shown gels are representative of at least 3 experiments. Right panel. Quantification using ImageJ software. Levels of Cytc, Cox2, Atp1 and Sdh2 are normalized to Por1p and expressed relative to WT. (C) Genetic ablation of REI1 or RPL6B in taz1Δ yeast preserves mitochondrial respiration. On the left are the rates of oxygen consumption from NADH (4 mM) alone (state 4), after further addition (150 μM) of ADP (state 3) or CCCP (4 μM) (uncoupled respiration). The data are expressed in % of WT state 4 respiration (mean ± s.d, n=4). On the right are the oxygen consumption rates from electrons delivered directly to complex IV by ascorbate 12.5 mM/TMPD 1.4 mM in the presence of CCCP. Data are expressed relative to the WT (mean ± s.d, n=4). (D) Mitochondrial respiration is preserved in taz1Δ yeast grown in the presence of 10 nM CHX. NADH was used as the electron donor, as described in panel C (n=4). (E) ATP synthesis was measured using NADH as a respiratory substrate in the presence of 1 mM ADP. Data are expressed as mean ± s.d. (n=4) relative to the WT. The data for WT and taz1Δ strains were reported previously [60].

60. de Taffin de Tilques M, Tribouillard-Tanvier D, Tetaud E, Testet E, di Rago JP, Lasserre JP (2017). Overexpression of mitochondrial oxodicarboxylate carrier (ODC1) preserves oxidative phosphorylation in a yeast model of Barth syndrome. Dis Model Mech 10(4): 439-450.

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