Ribosome stalling along TIM50 mRNA under eIF5A deficiency favors translation downregulation of mitoproteins. (A) Scheme showing ribosome stalling along TIM50 mRNA and Tim50 translocase receptor protein levels (in blue) when expressing different versions of TIM50. P indicates a proline-encoding codon. The figure was processed using BioRender (RRID:SCR_018361). (B) Wild-type and tif51A-1 strains expressing Tim50, Tim50∆Pro or both were cultured in SGal medium until reaching post-diauxic phase at 25°C and transferred to 37°C for 4 h. mRNA relative levels from PDR5 were determined by RT-qPCR. (C) Wild-type and tif51A-1 strains harboring Tim50, Tim50∆Pro, or both and expressing PDR5p-nLuc were cultured in YPD until the early exponential phase and then transferred to 25°C or 37°C for 4 h. The luminescence levels generated by the nLuc after the addition of the furimazine substrate were measured along time and the protein was quantified after 10 min. (D) Wild-type and tif51A-1 strains harboring Tim50, Tim50∆Pro, or both and expressing Cyc1-nLuc (left) or Cox5a-nLuc (right) were cultured as in C. Luminescence levels were measured as in C. Data information: In B–D, 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. The asterisks located above the columns of the graph represent a comparison relative to the wild-type strain. The asterisks found at the top of the bars additionally indicate comparisons between the different Tim50 versions within the same strain. n.s means no significant differences.