Figure S4.
eIF5A deficiency provokes ribosome stalling in TIM50 and other mitoprotein mRNAs, without affecting TIM50 mRNA levels nor its mitochondrial localization but reducing Tim50 protein levels. (A) Amino acid sequence alignment of genes encoding Tim50 in Mus musculus, Homo sapiens, Schizosaccharomyces pombe, Schizosaccharomyces jamonicus, Candida lipolytica, Candida albicans, Saccharomyces cerevisiae, and Candida glabrata. Residues with an asterisk are conserved in all organisms. The region containing the seven consecutive proline residues is highly conserved across yeast species and is marked by a box. (B) Wild-type and tif51A-1 strains containing genomic tagged Tim50-HA were cultured in SGal until post-diauxic phase at 25°C and transferred to 25°C or 37°C for 4 h. Tim50 protein levels were determined by western blotting (left) and quantified (middle). G6pdh levels were used as loading control. A representative image is shown from three independent experiments. TIM50 mRNA relative levels were determined by RT-qPCR (right). (C) Wild-type, tif51A-1, and tif51A-3 strains harboring a FLAG-TIM50-GFP plasmid were cultured in SRaf-URA at 25°C until early exponential phase, transferred to 25°C and 37°C for 2 h, and then transferred to SGal-URA at 25°C and 37°C for three additional hours. Tim50 protein levels were determined by western blotting using a FLAG antibody (left) and quantified (middle). G6pdh levels were used as loading control. A representative image is shown from three independent experiments. TIM50 mRNA relative levels were determined by RT-qPCR (right). (D) Wild-type strain and tif51A-1 expressing the wild-type Tim50 (left) or Tim50∆7Pro (right) nLuc constructs were cultured in YPD at 25°C or 37°C for 4 h. One hour after addition of doxycycline to induce nLuc expression, nLuc mRNA relative levels were determined by RT-qPCR. (E) Fraction of ribosome reads of various lengths along YTA12 (left) and MSS51 (right) transcripts in Schuller et al. (2017) ribosome profiling libraries. (F) Wild-type strain and tif51A-1 were cultured as in B and then subjected to phase contrast and fluorescence microscopy. Mitochondria were visualized by Su9-mCherry and TIM50 mRNAs were visualized by the single molecule MS2 tag system. A representative image is shown from three independent experiments. Scale bar, 4 μm. (G) Wild-type strain and tif51A-1 expressing Tim50-GFP and Su9-mCherry were cultured in SGal medium at 25°C until reaching post-diauxic phase and subjected to fluorescence microscopy. A representative image is shown from three independent experiments. Scale bar, 4 μm. Arrows indicate TIM50 mRNA. (H) Quantification of Tim50 and Cyc1 aggregates co-localizing with Hsp104 (data from three biological experiments with at least 150 cells quantified). Data obtained from experiments in Fig. 3 I and Fig. 4 J. Data information: In B–D and H, 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 < 0.01, ***P < 0.001. The asterisks located above the columns of the graph represent a comparison between different temperatures within the same strain. The asterisks found at the top of the bars additionally indicate comparisons between the indicated strains at the same treatment condition. n.s means no significant differences. Source data are available for this figure: SourceData FS4.

eIF5A deficiency provokes ribosome stalling in TIM50 and other mitoprotein mRNAs, without affecting TIM50 mRNA levels nor its mitochondrial localization but reducing Tim50 protein levels. (A) Amino acid sequence alignment of genes encoding Tim50 in Mus musculus, Homo sapiens, Schizosaccharomyces pombe, Schizosaccharomyces jamonicus, Candida lipolytica, Candida albicans, Saccharomyces cerevisiae, and Candida glabrata. Residues with an asterisk are conserved in all organisms. The region containing the seven consecutive proline residues is highly conserved across yeast species and is marked by a box. (B) Wild-type and tif51A-1 strains containing genomic tagged Tim50-HA were cultured in SGal until post-diauxic phase at 25°C and transferred to 25°C or 37°C for 4 h. Tim50 protein levels were determined by western blotting (left) and quantified (middle). G6pdh levels were used as loading control. A representative image is shown from three independent experiments. TIM50 mRNA relative levels were determined by RT-qPCR (right). (C) Wild-type, tif51A-1, and tif51A-3 strains harboring a FLAG-TIM50-GFP plasmid were cultured in SRaf-URA at 25°C until early exponential phase, transferred to 25°C and 37°C for 2 h, and then transferred to SGal-URA at 25°C and 37°C for three additional hours. Tim50 protein levels were determined by western blotting using a FLAG antibody (left) and quantified (middle). G6pdh levels were used as loading control. A representative image is shown from three independent experiments. TIM50 mRNA relative levels were determined by RT-qPCR (right). (D) Wild-type strain and tif51A-1 expressing the wild-type Tim50 (left) or Tim50∆7Pro (right) nLuc constructs were cultured in YPD at 25°C or 37°C for 4 h. One hour after addition of doxycycline to induce nLuc expression, nLuc mRNA relative levels were determined by RT-qPCR. (E) Fraction of ribosome reads of various lengths along YTA12 (left) and MSS51 (right) transcripts in Schuller et al. (2017) ribosome profiling libraries. (F) Wild-type strain and tif51A-1 were cultured as in B and then subjected to phase contrast and fluorescence microscopy. Mitochondria were visualized by Su9-mCherry and TIM50 mRNAs were visualized by the single molecule MS2 tag system. A representative image is shown from three independent experiments. Scale bar, 4 μm. (G) Wild-type strain and tif51A-1 expressing Tim50-GFP and Su9-mCherry were cultured in SGal medium at 25°C until reaching post-diauxic phase and subjected to fluorescence microscopy. A representative image is shown from three independent experiments. Scale bar, 4 μm. Arrows indicate TIM50 mRNA. (H) Quantification of Tim50 and Cyc1 aggregates co-localizing with Hsp104 (data from three biological experiments with at least 150 cells quantified). Data obtained from experiments in Fig. 3 I and Fig. 4 J. Data information: In B–D and H, 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 < 0.01, ***P < 0.001. The asterisks located above the columns of the graph represent a comparison between different temperatures within the same strain. The asterisks found at the top of the bars additionally indicate comparisons between the indicated strains at the same treatment condition. n.s means no significant differences. Source data are available for this figure: SourceData FS4.

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