Figure 2.

The interaction between Isu1–GST and Yfh1 is dependent on the concentrations of Isu1 and Yfh1. (A) Wild-type (WT) and Gal–ISU1/Δisu2 cells were grown on synthetic minimal media supplemented with galactose (Gal) or glucose (D). Mitochondria were isolated and analysed by immunostaining for Isu1, porin (Por2) and Mge1. (B,C) Isu1–GST was overproduced in Gal–ISU1/Δisu2 cells containing a plasmid (pCM182–YFH1) that carries YFH1 under the control of the TetO2 promoter. In this strain, levels of native Isu1 (without GST) are downregulated by growth in the presence of glucose, and the overexpression of YFH1 is blocked by the addition of 5 μg ml⁻¹ doxycycline. Mitochondria were isolated from cells cultivated on synthetic minimal media supplemented with galactose (ISU1 up) or glucose (ISU1 down) in the absence (YFH1 up) or presence (YFH1 down) of doxycycline, as indicated. Further analysis was carried out as described for Fig. 1. Bound, glutathione-S-transferase (GST)-affinity purified protein; In, input mitochondrial extract (10% of total); NB, non-bound fraction (10% of total).