Figure 8.

Conserved cysteine residues of Aim32 are important for assembly/stabilization of the TIM22 and TIM23 complexes.A, growth analysis of the indicated yeast strains after 2 to 3 days of incubation at 30 °C and 37 °C on different media as in Figure 3. Strains include WT and Δaim32 transformed with a plasmid that contains AIM32 with a single cysteine mutation (C213S or C222S) and a C-terminal FLAG tag or the empty vector (Vec). Data shown are representative of three independent experiments. B, as in Figure 5A, levels of mitochondrial proteins were analyzed by immunoblotting with antibodies against the indicated proteins. Strains are from panel A and also include AIM32 with a C-terminal FLAG tag (pAIM32). C, as in Figure 5B, 75-μg mitochondria from the indicated strains were solubilized in 1% (wt/vol) digitonin and subjected to blue native (BN)-PAGE (4–16% acrylamide) to detect TIM22, and D. TIM23^SORT, and the TIM23^CORE complexes. For the Yme1 complex, a 3 to 12% acrylamide BN-PAGE was used. Asterisks (∗ and ∗∗) indicate subcomplexes of Tim22 and Tim23. The box indicates the nonspecific band.