Figure 4.
eIF5A depletion causes TIM50 polyproline ribosome stalling, decreased Tim50 protein levels, and mislocalization. (A) Scheme showing Tim50 protein sequence. Proline amino acids (P) in the proline-rich region are typed in red color. (B) Scheme showing the two different nLuc versions used in this study. Proline numbers are shown in magenta. (C and D) Wild-type strain (left) and tif51A-1 (middle) expressing the wild-type Tim50 (C) or Tim50∆7Pro (D) were cultured in YPD until early exponential phase and then transferred to 25°C or 37°C for 4 h. After the addition of doxycycline to induce luciferase expression, the luminescence levels generated by nLuc after the addition of the furimazine substrate were measured along time (expressed as relative luciferase units, RLU). A representative experiment is shown. Quantification of the Tim50 protein levels is shown on the right. (E) Full protein synthesis time was calculated for wild-type strain and tif51A-1 expressing the wild-type Tim50 (left) or Tim50∆7Pro (right). (F) Fraction of ribosome reads of various lengths along TIM50 transcript in Schuller et al. (2017) ribosome profiling libraries. (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, transferred to 37°C for 4 h, and subjected to fluorescence microscopy. (H) Quantification of cells with Tim50 aggregates at 37°C is shown. (I) Wild-type strain and tif51A-1 expressing Tim50-GFP and Su9-mCherry were cultured as in G and incubated for 30 min with DAPI prior microscopy to stain the nuclei. (J) Wild-type strain and tif51A-1 expressing Tim50-GFP and Hsp104-RFP were cultured as in G. (G, I, and J) A representative image is shown from three independent experiments. Arrows indicate Tim50 cytosolic aggregates. Scale bar, 4 μm. Data information: In C–E and H Results are presented as mean ± SD from at least 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 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.

eIF5A depletion causes TIM50 polyproline ribosome stalling, decreased Tim50 protein levels, and mislocalization. (A) Scheme showing Tim50 protein sequence. Proline amino acids (P) in the proline-rich region are typed in red color. (B) Scheme showing the two different nLuc versions used in this study. Proline numbers are shown in magenta. (C and D) Wild-type strain (left) and tif51A-1 (middle) expressing the wild-type Tim50 (C) or Tim50∆7Pro (D) were cultured in YPD until early exponential phase and then transferred to 25°C or 37°C for 4 h. After the addition of doxycycline to induce luciferase expression, the luminescence levels generated by nLuc after the addition of the furimazine substrate were measured along time (expressed as relative luciferase units, RLU). A representative experiment is shown. Quantification of the Tim50 protein levels is shown on the right. (E) Full protein synthesis time was calculated for wild-type strain and tif51A-1 expressing the wild-type Tim50 (left) or Tim50∆7Pro (right). (F) Fraction of ribosome reads of various lengths along TIM50 transcript in Schuller et al. (2017) ribosome profiling libraries. (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, transferred to 37°C for 4 h, and subjected to fluorescence microscopy. (H) Quantification of cells with Tim50 aggregates at 37°C is shown. (I) Wild-type strain and tif51A-1 expressing Tim50-GFP and Su9-mCherry were cultured as in G and incubated for 30 min with DAPI prior microscopy to stain the nuclei. (J) Wild-type strain and tif51A-1 expressing Tim50-GFP and Hsp104-RFP were cultured as in G. (G, I, and J) A representative image is shown from three independent experiments. Arrows indicate Tim50 cytosolic aggregates. Scale bar, 4 μm. Data information: In C–E and H Results are presented as mean ± SD from at least 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 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.

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