Fig. 1.

Mitochondrial inhibitors differentially affect stability of the TIM protein. Drosophila S2R + cells were acutely treated with vehicle or specific respiratory chain inhibitors in the dark, followed by continued darkness or light exposure. (A,B) Inhibitors of complex III (antimycin A, Ant; myxothyzol, Myx) and V (oligomycin, Oli) block light-dependent TIM degradation, whereas complex I inhibitor rotenone (Rot) promotes TIM degradation. No discernible effects were observed for complex II inhibitors TTFA, atpenin (Atp), oxaloacetate (Oxa), and complex IV inhibitor sodium cyanide (NaCn). Representative Western blots are shown in (A) and intensity of the TIM and MAPK loading control bands are quantified in (B). (C,D) Effect of rotenone on TIM stability requires CRY. In cells transfected with the mutant construct cry^b, rotenone has minimal effect on TIM degradation. In contrast, rotenone promotes TIM degradation in the presence of CRY. Representative Western blots are shown in (C) and intensity of the TIM and b-GAL loading control bands are quantified in (D). (E,F) In cells expressing wildtype CRY, the effect of rotenone (2 μM) on TIM stability is partially rescued by antimycin A (2 μM); In contrast, TIM is destabilized in cells expressing a CRY that lacks its C-terminus (CRY^m), and antimycin A does not block the effect of CRY^m on TIM. Black filled bars denote darkness, and open bars denote light conditions in B and D. Error bar denotes standard error of the mean, n > 3. Asterisk denotes significant difference (p < 0.05) by Student’s t-test. Different letters above the bars denote significant differences between bars on either the left or the right side of panel F (one-way ANOVA and post hoc Tukey test, p < 0.05). Full-length blots are presented in Supplementary Figures S2, S8, S9.