eIF5A deficiency generates mitoCPR stress and mislocalization of mitoproteins. (A) Wild-type strain and tif51A-1 were cultured in SGal medium at 25°C until reaching post-diauxic phase and then transferred to 25°C or 37°C for 4 h. mRNA relative levels from mitoCPR genes were determined by RT-qPCR. (B) Wild-type strain and tif51A-1 expressing Pdr5-GFP were cultured as in A and then subjected to fluorescence microscopy. (C) Quantification of Pdr5-GFP fluorescent signal from at least 150 cells. (D–G) Wild-type strain and tif51A-1 expressing Tom70-GFP (D), Yta12-GFP (E), Cyc1-GFP (F), or Ilv2-GFP (G) and Su9-mCherry were cultured as in A and subjected to fluorescence microscopy. Scale bar, 4 μm. (E) Quantification of cells with Yta12 aggregates at 37°C is shown (right). (H) Wild-type strain and tif51A-1 expressing Cyc1-GFP and Su9-mCherry were cultured as in A and incubated for 30 min with DAPI prior microscopy to stain the nuclei. Scale bar, 4 μm. (I) Wild-type strain and tif51A-1 expressing Cyc1-GFP and Hsp104-RFP were cultured as in A and subjected to fluorescence microscopy. Scale bar, 4 μm. (B and D–I) A representative image is shown from three independent experiments. Arrows in E–H indicate mitoprotein cytosolic aggregates. Data information: In A, C, and E, results are presented as mean ± SD from three independent experiments. The statistical significance was measured by using a two-tailed paired Student’s t test. *P < 0.05, ***P < 0.001. n.s means no significant differences.