The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells
- University of Cambridge, United Kingdom
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, United Kingdom
- Medical Research Council Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 1 supplement
A CRISPR-Cas9-based genome-wide screen implicates the ribosomal P-stalk in ISR induction.
(A) Overlay plot of the fluorescence signal from wildtype (WT) or GCN2-ablated Eif2ak4∆ mutant HeLa cells with an ISR sensitive CHOP::GFP reporter (horizontal axis Ex: 488 nm/ Em530 ± 30 nm) and a …
Figure 1—figure supplement 1
Poor clonogenic potential of stressed HeLa compared with CHO cells.
(A) Photograph of crystal violet stained plates of cultured and CHO cells following exposure to the indicated concentration of histidinol (in mM, for 20 hr), starvation of lysine and arginine (-KR, …
Figure 2
CRISPR-Cas9-based P-stalk lesions impair CHOP::GFP induction upon histidinol treatment or amino acid starvation.
(A) Flow cytometry analysis of the ISR-inducible CHOP::GFP and the UPR inducible XBP1::mCherry reporters from untreated (UT red), histidinol-treated (0.5 mM, HD, blue), thapsigargin-treated (1 mM, …
Figure 3 with 1 supplement
Impaired translational control, GCN2 activation, and ISR induction in amino acid starved P-stalk mutant cells.
(A) Overlaid A260 traces from 10–50% sucrose gradients loaded with cytoplasmic extracts from wildtype and the indicated mutant cells left untreated (UT, black) or starved for lysine and arginine …
Figure 3—figure supplement 1
Impaired translational control, GCN2 activation, and ISR induction in amino acid starved P-stalk mutant cells (reporting on reproducibility of the observations shown in Figure 3).
(A) GCN2 immunoblot of extracts from wildtype (WT), compound P-stalk Rplp0/Rplp1m29-132 double mutant cells, or GCN2-ablated Eif2ak4Δ mutant cells, untreated or starved of lysine and arginine (-KR) …
Figure 4
Defective ribosome association of P1 and P2 in mutant CHO cells.
(A) Immunoblot of cytoplasmic extracts from untreated or histidinol-treated (HD, 0.5 mM) wildtype and the indicated mutant CHO cells. The polyclonal serum (anti uL101-200) recognizes the N-terminal …
Figure 5 with 1 supplement
P-stalk lesions impair ribosome stimulation of eIF2α directed GCN2 kinase activity.
(A) Immunoblot of eIF2α from in vitro phosphorylation reactions resolved by phos-tag SDS-PAGE. The concentration of purified GCN2 kinase, ribosomes, and the genotype of the ribosomes is indicated …
Figure 5—figure supplement 1
P-stalk lesions (reproducibly) impair ribosome stimulation of eIF2α directed GCN2 kinase activity.
(A) Immunoblot of eIF2α from in vitro phosphorylation reactions resolved by phos-tag SDS-PAGE, as in Figure 5A but utilizing different preparation of wildtype and Rplp0/Rplp1m29-132 compound mutant …
Figure 6 with 1 supplement
Ribosome activation alters GCN2 enzyme kinetics.
(A) Coomassie-stained phos-tag PAGE of eIF2α from in vitro phosphorylation reactions with purified GCN2 or the cytosolic kinase domain of PERK, in presence or absence of wildtype ribosomes. (B) …
Figure 6—figure supplement 1
Ribosome activation alters GCN2 enzyme kinetics.
(A) Immunoblot of eIF2α from in vitro phosphorylation reactions with escalating concentration of eIF2α substrate and ribosomes from cells of the indicated genotype (added at 30 nM). Aliquots of each …
Figure 7
A model for ribosome state-dependent GCN2 activation by the P-stalk.
(A) In amino acid replete cells, charged tRNAs and elongation factors eEF1A and eEF2 cycle through the GTPase associated centre repressing the P-stalk’s latent ability to activate GCN2. Repression …
Additional files
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Supplementary file 1
Key resources table.
- https://cdn.elifesciences.org/articles/50149/elife-50149-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50149/elife-50149-transrepform-v2.docx
