Fig 1: eIF2α Phosphorylation Attenuates Translation of Mitochondrial RPs(A) Boxplots of Torin1-induced changes in translation efficiency for all mRNAs encoding CytoRPs and MitoRPs analyzed from Thoreen et al. (2012). ∗∗∗p < 0.001, as determined by an unpaired Wilcoxon test.(B) Representative polysome profiles from eIF2α S/S and eIF2α A/A MEFs at 0 and 2 h following treatment with 2.5 μg/mL Tm.(C) Mrpl12, Mrpl28, Mrps25, and Mrps15 mRNA abundance from heavy polysomal fractions (more than three ribosomes) shown in (B) assessed by qPCR. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, as determined by an unpaired Student t test. Representative results of at least three independent experiments are shown. Data are mean ± SEM (n = 3).See also Figure S6.
Fig 2: Translationally Downregulated Proteins following eIF2α Phosphorylation Are Long-Lived(A) Experimental design (top panel) to measure mitochondrial translation. 35S methionine/cysteine incorporation into newly translated mitochondrial proteins (encoded by mtDNA) following chloramphenicol (Cm; 50 μg/mL) or Tm (2.5 μg/mL) treatment of HEK293T cells for the indicated times. Cytoplasmic translation was inhibited using emetine (100 μg/mL). ATF4 was used as a positive control for the Tm treatment. Representative results of at least three independent experiments are shown.(B) Representative immunoblots showing the abundance of RPL4, RPS6, MRPL44, MRPL28, ATF4, and tubulin proteins at the indicated times after treatment of HeLa cells with 2.5 μg/mL Tm. ATF4 was used as a positive control for the Tm treatment.(C) Same as (B), but following 20 μg/mL cycloheximide (CHX) treatment. POMP (a short-lived protein) was used as a control for the CHX treatment.(D) Histogram showing the distribution of protein half-lives encoded by translationally down- or upregulated mRNAs (Down or Up) at 2 h following Tm injection.(E) Boxplot of half-lives of proteins encoded by the translationally down- or upregulated mRNAs (Down or Up), all 79 CytoRPs, or all 82 MitoRPs at 2 h following Tm injection.(F) Same as (E), but with exclusion of the RPs (Down w/o RPs).In (E) and (F), protein half-lives were analyzed from Sandoval et al. (2013). ∗∗p < 0.01 and ∗∗∗p < 0.001, as determined by an unpaired Wilcoxon test. See also Figures S8 and S9.
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