Deletion of the Tim50 polyproline stretch in the eIF5A mutant does not rescue mitochondrial respiration but cancels the mitoCPR response induction. (A) Growth of the wild-type, tif51A-1, wild-type Tim50∆Pro, and tif51A-1 Tim50∆Pro was tested in YPGly and YPD media at the indicated temperatures. (B–E) Wild-type, tif51A-1, wild-type Tim50∆Pro, and tif51A-1 Tim50∆Pro were cultured in SGal medium until reaching post-diauxic phase at 25°C, transferred to 37°C for 4 h, and subjected to fluorescence microscopy. A representative image from three independent experiments is shown. Scale bar, 4 μm (B and D). Cells were incubated for 30 min with Mitotracker prior to microscopy to quantify the mitochondrial membrane potential (B). Quantification of Mitotracker fluorescent signal from at least 150 cells (C). Quantification of cells presenting 0, 1, or 2 Tim50 aggregates at 37°C is shown (E). (F) Wild-type, tif51A-1, and tif51A-1 carrying Tim50∆Pro were cultured as in B. mRNA relative levels from mitoCPR genes were determined by RT-qPCR. Data information: In C, E, and F, results are presented as mean ± SD from three independent experiments. The statistical significance was measured by using a two-tailed Student’s t test. *P < 0.05, **P < 0.001, ***P < 0.001. n.s means no significant differences.