doi: 10.1038/s41586-020-2446-y.
Epub 2020 Jul 1.
Systematic quantitative analysis of ribosome inventory during nutrient stress
Affiliations
- PMID: 32612236
- PMCID: PMC7351614
- DOI: 10.1038/s41586-020-2446-y
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Systematic quantitative analysis of ribosome inventory during nutrient stress
Nature.
2020 Jul.
Abstract
Mammalian cells reorganize their proteomes in response to nutrient stress through translational suppression and degradative mechanisms using the proteasome and autophagy systems1,2. Ribosomes are central targets of this response, as they are responsible for translation and subject to lysosomal turnover during nutrient stress3-5. The abundance of ribosomal (r)-proteins (around 6% of the proteome; 107 copies per cell)6,7 and their high arginine and lysine content has led to the hypothesis that they are selectively used as a source of basic amino acids during nutrient stress through autophagy4,7. However, the relative contributions of translational and degradative mechanisms to the control of r-protein abundance during acute stress responses is poorly understood, as is the extent to which r-proteins are used to generate amino acids when specific building blocks are limited7. Here, we integrate quantitative global translatome and degradome proteomics8 with genetically encoded Ribo-Keima5 and Ribo-Halo reporters to interrogate r-protein homeostasis with and without active autophagy. In conditions of acute nutrient stress, cells strongly suppress the translation of r-proteins, but, notably, r-protein degradation occurs largely through non-autophagic pathways. Simultaneously, the decrease in r-protein abundance is compensated for by a reduced dilution of pre-existing ribosomes and a reduction in cell volume, thereby maintaining the density of ribosomes within single cells. Withdrawal of basic or hydrophobic amino acids induces translational repression without differential induction of ribophagy, indicating that ribophagy is not used to selectively produce basic amino acids during acute nutrient stress. We present a quantitative framework that describes the contributions of biosynthetic and degradative mechanisms to r-protein abundance and proteome remodelling in conditions of nutrient stress.
Conflict of interest statement
COMPETING INTEREST STATEMENT
The authors declare the following competing interests: J.W.H. is a founder and consultant for Caraway Therapeutics and a consultant for X-Chem, Inc.
Figures
a, Schematic of the ribosome analysis pipeline. b-d, (top panels) Volcano plots (−Log10 p-value versus Log2 ratio Tor1/UT) for 293T cells (n=8029 proteins), ATG7−/− (n=8373 proteins) or RB1CC1−/− (n=8332 proteins) (panel b), 293 cells (n=7531 proteins), ATG5−/− (n=7504 proteins) (panel c), or HCT116 cells (n=3779 proteins), ATG5−/− (n=3761 proteins) or RB1CC1−/− (n=3671 proteins) (panel d). n=3 (WT); 4 (Tor1) biologically independent samples. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 1. Green dots represent ribosomal proteins. Data for 293T cells is from. Histograms below the individual volcano plots show the mean ± SD of relative abundance of autophagy adaptors with or without nutrient deprivation. n=3 (WT) or n=4 (-AA and Tor1) biologically independent samples. U, untreated; A, -AAs; T, Tor1. Data for 293T cells was from. e, The relative abundance changes for proteins located in either the endoplasmic reticulum, Golgi, or the ribosome in 293T cells treated as in panel b are plotted as a violin plot (n=340, 349, 343, 340, 349, 343, 87, 89, 86, 87, 89, 86, 72, 75, 70, 72, 75, and 70 proteins, from left to right). Ribosomal protein abundance change is not affected by autophagy unlike other vesicular organelles. The violin curves represent the distribution and density of the indicated dataset (Center-line: median; Limits: minima and maxima). f, Plots of relative abundance of individual r-protein in 293 cells upon either 10h of AA withdrawal (left panel) or Tor1 treatment (right panel). 39 r-proteins with less than ±10% error range for every condition are plotted. Mean ±SD for n=3 (WT) or n=4 (-AA and Tor1) biologically independent samples. g, 293T cells with or without ATG7 or RB1CC1 were either left untreated, subjected to AA withdrawal (10h) or treated with Tor1 (10h) and whole cell extracts immunoblotted for the indicated proteins. h, HCT116 cells with or without ATG5 or RB1CC1 were treated as indicated, and whole cell extracts immunoblotted for the indicated proteins. i-k, Extracts from the indicated cells (30, 15, 7.5 or 3.75 μg) were immunoblotted with the indicated antibodies (panels i,j). The signal intensity for the indicated r-proteins as a function of quantity loaded was measured using Odyssey (panel k), showing no indication of signal saturation and no detectable difference between cells with or without active autophagy. Related to Fig. 1. The experiments shown in panels g-j were repeated more than three times independently and showed similar results. For gel source data, see Supplementary Fig. 1.
a, The Halotag7 protein (referred to here as “Halo”) was endogenously tagged at the C terminus of RPL29 or RPS3 as the indicated r-proteins contain solvent exposed C-termini and are located far from the peptide exit tunnel based on the structure of an 80S complex. (PDB: 5AJ0). b,c, Gene editing of HCT116, 293, and 293T cells using CRISPR-Cas9 to fuse Halo with the C-termini of RPS3 and RPL29. Homozygous incorporation of Halo was confirmed by genotyping (panel b). Extracts from the indicated cells were subjected to immunoblotting with RPS3 (panel c, left) or RPL29 (panel c, right). Protein translation efficiency of wild-type and Halo knock-in cells were compared using puromycin incorporation assay (panel c, bottom). d, Immunoblotting of WT or RPS3-Halo 293T cell lysates after nutrient stress, confirming no detectable difference between the two cell lines in response to mTOR inhibition. The full immunoblot is shown in Extended Data Fig. 9i. e-g, Halo-ligand titration assays with the indicated incubation time were performed using flow-cytometry analysis (panels e and f) or in-gel fluorescence analysis (panel g) for the labeling saturation. In panel f, background signal from free Halo-ligand was measured using WT HCT116 cells in comparison to RPS3-Halo, confirming that the free ligand does not contribute to the observed fluorescence signal. h, HCT116 RPS3-Halo cells were incubated with 250 nM Halo-TMR ligand for 1h, washed for indicated numbers by incubating cells in ligand free medium for 20 minute each time, followed by 17h prolonged incubation in ligand free medium. i, Extracts from the RPS3-Halo HCT116 cells (20, 15, 10 or 5 μg) treated with the indicated Halo ligands were subjected to in-gel fluorescence analysis. The fluorescence signal intensity of each lane was directly proportional to the loading amount. We noted that R110 fluorophore was excited by epi-green excitation (520–545 nm) and detected in 577–613 nm. We subtracted this bleed-through signal for TMR quantification. j, Measurement of the effect of Tor1 on cell size with HCT116 RPS3-Halo (left) and RPL29-Halo (right) using Coulter Principle-based cell measurements. k, Cell proliferation assay. HCT116 RPS3-Halo and RPL29-Halo cells were grown in rich medium or Tor1 (200 nM) containing medium for 12h, 16h, and 24h. The data estimates ~16h cell division rate for untreated cells, and ~24h cell division rate for Tor1 treated cells. Mean ±SD for n=4 biologically independent experiments. l, Ratio of pre-existing to newly synthesized RPL29-Halo per cell plotted against cell populations as a frequency histogram (left panel). Average from the triplicate experiments plotted as a bar graph (right panel). Pre-existing Ribo-Halo proteins in HCT116 RPL29-Halo cells were labeled with TMR-ligand (100 nM, 1h), followed by the thorough washing and addition of 50 nM Green-ligand (also called R110-ligand). The newly synthesized RPL29-Halo was chased for 8, 16, and 24 hours prior to flow-cytometry analysis. Error bars represent SD. See METHODS for details. m, Pre-existing RPL29-Halo proteins were labeled with TMR-ligand (100 nM, 1h) in HCT116 cells, and the newly synthesized RPL29-Halo in the presence or absence of Tor1 (200 nM) were labeled with Green-ligand. The ratio of R110 to TMR signals plotted against cell populations (left panel), and the mean values from the triplicate experiments of 8h, 16h, and 24h pulse chase plotted as a bar graph (right panel) are shown. Error bars represent SD. n, In-gel fluorescence images of the cell extracts treated as in panel m. The same gels were then transferred to PVDF membranes for immunoblotting measurement of total RPL29 level. n=3 biologically independent samples are shown. o, In-gel fluorescence images of the cell extracts from 293T RPS3-Halo or HCT116 RPS3-Halo cells using the labeling strategy in panel m. Relative synthesis of RPS3-Halo with or without Tor1 is plotted on the right. Mean for n=2 experiments. p, Live-cell imaging of HCT116 RPS3-Halo cells labeled with TMR (for pre-existing r-proteins) and Green (for newly synthesized r-proteins) ligands with or without Tor1 (200 nM, 24h). Scale bar = 20 μm. q, Live-cell imaging of HCT116 RPL29-Halo cells labeled with TMR (for pre-existing r-proteins) and Green (for newly synthesized r-proteins) ligands with or without Tor1 (200 nM, 14h). Scale bar = 20 μm. r, The indicated cells were left untreated or incubated with Tor1 for 8h prior to immunoblotting with the indicated antibodies. Related to Fig. 2. The experiments in panels d, j, and p-r were repeated three times independently and showed the similar results, and panels b-c and e-i were performed once. For gel source data, see Supplementary Fig. 1.
a,b, Histogram of normalized TMR signal in RPS3-Halo (panel a) and RPL29-Halo (panel b) HCT116 cells with or without ATG8 conjugation (ATG7 for RPS3-Halo and ATG5 for RPL29-Halo) or RB1CC1 incubated with or without 200 nM Tor1 for 14h, followed by 1h TMR ligand treatment and flow-cytometry analysis. >3×105 and >4×103 cells were analyzed, respectively. c,d, Mean of the triplicate data from cells treated as in panel a and b are plotted, respectively. Error bars represent SD. e, Effect of Tor1 treatment on cell size in HCT116 RPL29-Halo WT, ATG5−/− and RB1CC1−/− cells, as measured using Coulter Principle-based cell measurements. Mean ± SD of the triplicate data is plotted. f,g, Ratio of pre-existing (red) to newly synthesized (green) r-proteins per cell plotted against cell populations as a frequency histogram for RPS3-Halo (panel f) or RPL29-Halo (panel g) HCT116 cells with or without ATG5/7 or RB1CC1 based on the labeling scheme in Fig. 2f. h, Quantification of relative amounts of pre-existing and newly synthesized r-proteins from data in panels f and g. Mean ± SD, n=3 biologically independent experiments. i,j, HCT116 RPS3-Halo cells with or without ATG7 or RB1CC1 were left untreated or treated with Tor1 for 14h (panel i) and 24h (panel j) using the Halo tagging scheme in Fig. 2f. Extracts were subjected to SDS-PAGE and in-gel fluorescence analysis, followed by immunoblotting with the indicated antibodies. k,l, Quantification of relative amounts of pre-existing and newly synthesized r-proteins from data in panels i and j. Mean ± SD, n=3 biologically independent experiments. m, Live-cell imaging of HCT116 RPL29-Halo cells with indicated genotypes labeled with TMR (for pre-existing r-proteins) and Green (for newly synthesized r-proteins) ligands with or without Tor1 (200 nM, 14h). Scale bar = 20 μm. n, An example of gating strategy used for flow-cytometry analysis. Green and Red only control experiments are shown at the bottom. Experiments in panels m and n were repeated more than three times independently with similar results. For gel source data, see Supplementary Fig. 1.
a,b, Total AHA incorporation levels with or without prior Methionine starvation (30 min) were compared using 293T cells grown in Met or AHA (250 μM) for the indicated duration, followed by click with TMR alkyne and in-gel fluorescence analysis (panel a). The quantification of duplicate experiments is shown in panel b. c,d, 293T cells were grown in media with Met (250 μM) or the indicated concentration of AHA for the indicated time periods. Extracts were clicked with TMR and subjected to SDS-PAGE prior to Coomassie staining or in-gel TMR fluorescence analysis (panel c). TMR intensity was quantified in panel d. e,f, 293T cells grown in AHA (250 μM) with or without To1 for the indicated time periods and extracts clicked with TMR prior to processing as in panel c. The effect of Tor1 on TMR fluorescence is quantified in panel f. g, 293T cells were incubated with or without AA withdrawal or Tor1 containing media in the presence of Met or AHA (250 μM each), as in Fig. 3c. Cell extracts were subjected to SDS-PAGE followed by immunoblotting. Three biologically independent samples are shown in the same blot. h, TMR signals in Fig. 3c. were quantified as described in METHODS and plotted in top panel, and the relative TMT signal of the total biotinylated proteome in Fig. 3d. is plotted in bottom panel. Centre data are mean ±SD. n=1, 3, 3 and 3 biologically independent samples, from left to right for top and bottom panels. i, Translatome analysis. 293T cells were carried through the workflow in Fig. 3b with AA withdrawal and extracts clicked with biotin prior to enrichment on streptavidin and TMT-based proteomics. Plot of −Log10 p-value versus Log2 ratio -AA/untreated is shown for n=8285 proteins. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 2. n=3 (UT; -AA) biologically independent samples. j, Translatome plot of −Log10 p-value versus Log2 ratio -AA/Tor1 is shown (n=8285 proteins). r-proteins skewed to the right side of the volcano plot indicates that Tor1 suppresses the translation of r-proteins more strongly than -AA, unlike the majority of the proteome. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 2. n=3 (-AA; Tor1) biologically independent samples. k-o, The relative abundance of all quantified biotinylated proteins are shown in panel k. Proteins in individual groups are shown in panels l-o. l: representative proteins showing over 2-fold more reduction in translation than the average translation in both Tor1 and -AA conditions, m: proteins with over 2-fold less reduction in translation than the average translation in both Tor1 and -AA conditions, n: proteins showing 2-fold less reduction only in Tor1 condition, with p-value (Supplementary Table 2) between Tor1 and -AA was less than 0.05. o: proteins showing 2-fold less reduction only in -AA condition, with p-value (Supplementary Table 2) between Tor1 and -AA was less than 0.05. n = 3 biologically independent samples per conditions (UT; -AA; Tor1). Centre data are mean ±S.E.M. p-r, 293T cells lacking either ATG7 or RB1CC1 were subjected to AA withdrawal or Tor1 treatment for 3h in the presence of 250 μM Met or AHA. Cell extracts were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies (panel p) or clicked with TMR and in-gel fluorescence analysis (panel q). TMR signals were quantified as described in METHODS (panel r). Centre data are mean ±SD. n=1, 3, 3 and 3 biologically independent samples, from left to right. s, Correlation plot (Log2 ratio of treated/untreated) for the translatome upon either Tor1 treatment or AA withdrawal. Related to Fig. 3. Experiments in c-f were performed once and p was performed three times independently with similar results. For gel source data, see Supplementary Fig. 1.
a, Extracts from cells as described in Fig. 3g were subjected to immunoblotting with the indicated antibodies to demonstrate suppression of the mTOR activity by Tor1. b, Biotinylated extracts as described in Fig. 3g were subjected to immunoblotting with a fluorescent streptavidin conjugant, showing the pre-existing proteome (Streptavidin-IRdye) versus the total proteome (Revert total protein stain) in the lysates. n=3 biologically independent samples are shown in a and b. c, Volcano plots (−Log10 p-value versus Log2 Tor1/UT 12h) for 293T WT (n=8304 proteins), ATG7−/− (n=8319 proteins), and RB1CC1−/− (n=8590 proteins) cells as in Fig. 3i, but with pattern 1 proteins in Fig. 3h colored as red dots, pattern 2 proteins colored as green dots, and pattern 3 proteins colored as blue dots. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 3. n=3 (UT; Tor1) biologically independent samples. d, Volcano plots as in panel c, but with autophagy related proteins colored as indicated. Autophagy adaptors dramatically degraded upon Tor1 treatment only in WT cells are shown in the circle. e, Plots of individual ratio for AHA-labeled ribosomal proteins employing the protocol in Fig. 3g in WT, ATG7−/−, or RB1CC1−/− 293T cells. r-proteins with STDEV < 0.3 for every condition are selected (n=58 r-proteins). Centre data are mean ±SD from 3 biologically independent samples for each condition. f, Relative turnover rates for individual r-proteins from panel e, with or without mTOR inhibition have a correlation of R2~0.7 (n=58 r-proteins). Grey dotted lines are 95% confidence intervals of the best-fit line (solid black line) result from a simple linear regression analysis. g, Volcano plots as in panel d, but with ER resident proteins colored in purple. h,i, Individual ratio for n=326 AHA-labeled ER membrane resident proteins employing the protocol in Fig. 3g in WT, ATG7−/−, or RB1CC1−/− 293T cells (panel h) and plots for all individual ER resident proteins data points used in h (panel i). Centre data are mean ±SEM. Related to Fig. 3. For gel source data, see Supplementary Fig. 1.
a, Schematic of AHA-based degradomics time-course to examine r-protein turnover during nutrient stress with or without functional autophagy. b, Lysates from 293T cells treated as in panel a, were subjected to SDS-PAGE followed by immunoblotting to confirm proper mTOR inhibition. c, Cell extracts were reacted with TMR alkyne for click reaction followed by in-gel fluorescence analysis. d, Click reaction yield across the replicates was confirmed indirectly. Briefly, cell extracts treated as in panel a, were clicked with biotin alkyne followed by streptavidin capture. The proteins in the flow-though was precipitated, re-suspended in 2% SDS, then clicked with TMR-alkyne. e, Patterns of protein turnover, as described in the main Fig. 3h, in the time-course experiment. mean ±SEM. Proteins analyzed (n=4 top; n=6 middle and bottom) are shown below. f, Volcano plots for the indicated time (−Log10 p-value versus Log2 Tor1/UT) in 293T WT (n=3334 proteins), ATG7−/− (n=3351 proteins), and RB1CC1−/− (n=3375 proteins) cells. r-proteins, red, green or blue. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 3. g, A boxplot of the individual ratio for AHA-labeled ribosomal proteins employing the protocol in panel a (center line, median; box limits correspond to the first and third quartiles; whiskers, 10–90 percentiles range). n=48 r-proteins that were quantified across WT, ATG7−/−, and RB1CC1−/− 293T cells. two-sided t-test, p = 0.1514, 0.2818, 0.0005, 0.000016, 0.000051, 0.0005, 0.000001, 0.000020, 0.0105 from left to right) ns=non-significant,
*P<0.1,
***P<0.001,
****P<0.0001. Experiments in b-d were replicated twice independently and showed similar results. For gel source data, see Supplementary Fig. 1.
a, Contribution of cytoplasmic and nuclear partitioning to net ribosome balance upon nutrient stress. b,c, Optimization of the nuclear and cytosolic fraction partitioning method using 293T cells. Side by side comparison of four previously published methods was performed. Surfactant used in method1: 1% Triton, method 2: 0.1% Triton, method 3: 0.1% NP40, method 4: 0.05% NP40. For more details on method 1–4, see METHOD. Method 2 and 3 were further compared in panel c. 1: ctrl, 2: Leptomycin B (20 nM, 16h), 3: MgCl2 added in lysis buffer, 4: 2+3, 5: ctrl, 6: Leptomycin B (20 nM, 16h), 7: MgCl2 added in lysis buffer, 8: additional pipetting. d, Effect of the centrifugal velocity and duration on nuclear-cytosol partitioning is shown. 1: 13K rpm, 10sec, 2: 10K rpm, 10sec, 3: 7K rpm, 10sec, 4: 7K rpm, 30sec, 5: 5K rpm, 60sec, 6: 5K rpm, 180sec, 7: 3K rpm, 60sec, 8: 3K rpm, 180sec. e,f, Lysates collected after Tor1 treatment for 0h, 1h or 3h subjected to the optimized nuclear-cytosol partitioning, followed by immunoblotting against the indicated antibodies (panel e). Quantification measured by Odyssey shown in panel f. g, Scheme depicting strategy for quantitative analysis of changes in nuclear and cytosolic protein abundance in 293T cells in response to short period (3h) of AA withdrawal. h,i, Biochemical characterization of nuclear and cytosolic 293T cell fractions in response to AA withdrawal. Extracts (15 μg of cytosol and nuclei) were separated by SDS-PAGE and immunoblots probed with the indicated antibodies (see METHODS). j,k, Volcano plots (−Log10 p-value versus Log2 -AA/UT) for nuclear (j) or cytosolic (k) proteins (n=9193 proteins) quantified by TMT-based proteomics. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 4. Nuclear fraction (j) n=3 (UT; -AA), cytosolic fraction (k) n=2 (UT); n=3 (-AA) biologically independent samples. l, Relative RPS6, FOXK1, FOXK2, and CAD phospho-peptides abundance quantified by TMT-based proteomics confirming strong inhibition of mTOR by Tor1. Centre data are mean ±SD. n=2 for UT RPS6 and CAD, and 3 for the rest. m,n, Relative abundance of proteins that translocate either from cytosol to nucleus (panel m) or from nucleus to cytosol (panel n) after 3h AA withdrawal, including proteins linked with nutrient dependent transcription (TFEB, MITF, TFE3 – accumulating in the nucleus), and proteins involved in ribosome assembly (PWP1, SDAD1, NVL – exported from the nucleus to the cytosol). Centre data are mean ±SD. n=3, 2, 3 and 3 biologically independent samples, from left to right for each indicated proteins. o,p, 293T cells were treated with -AA medium for the indicated time period, partitioned into nuclear and cytosolic fractions, followed by immunoblotting against the indicated antibodies (panel o). Odyssey quantification shown in panel p. Experiments shown in b-d were performed once, e,h,i were performed more than three times independently with similar results, and o was performed twice with similar results. For gel source data, see Supplementary Fig. 1.
a, Relative abundance of individual r-proteins from the 60S subunit (left) or 40S subunit (right) with the cytosol fraction in grey and the nuclear fraction in blue. (less than ±10% error range for every replicate) Individual r-proteins that are thought to assemble onto the ribosome either late in the assembly process or specifically in the cytosol are indicated in red font. mean ±SD. n=2 for cytosolic fraction and 3 for nuclear fraction as shown in Extended Data Fig. 7g. b,c, Abundance of nuclear and cytosolic r-proteins after AA withdrawal (3h). 60S subunits are on top (n=38), and 40S subunits are at the bottom (n=26). Right panels indicate the relative r-protein abundance change normalized by UT of cytosolic or nuclear fraction. mean ±SEM. d, -AA/UT ratio of individual r-proteins from both nuclear and cytosolic fractions collected after 3h AA starvation indicates heterogenous distribution with RPS7 most strongly down regulated. n=64, mean ±SEM. e, Lysates from HEK293 RPS3-Keima cells after the indicated nutrient stress were immunoblotted against anti-Keima antibody (top). Abundance ratio of the processed Keima to the intact Keima measured by Odyssey is plotted (bottom). f, Flow-cytometry analysis of HEK293 RPS3-Keima cells to obtain normalization factors. To achieve a condition in which cells have 0% of the ribosomes in the lysosome, RPS3-Keima cells were treated with SAR405 for 10h and collected in pH7.2 FACS buffer. To achieve a theoretical condition in which 100% ribosomes are present in the lysosome, the cells were incubated in pH4.5 FACS buffer containing 0.1% Triton-X. We used the 561/488 ratio from these two measurements to calculate the % lysosomal ribosomes in panels g and h. n=1742 cells for each. g,h, HEK293 RPS3-Keima cells were left untreated or treated with Tor1 (200 nM) in the presence or absence of SAR405 (1 μM) for 12 hours. 561 nm ex to 488 nm ex Keima signal was measured by flow-cytometry and plotted as either a frequency histogram (panel g, n=1742 cells for each) or a bar graph (panel h, n=3 biologically independent samples, mean ±SD). i, TMT-based quantification of endogenous RPS3 abundance in 293T WT, ATG7−/−, and RB1CC1−/− cells treated and processed as in Fig. 3g. n=3 biologically independent samples. mean ±SD. Experiments in e,g,h were repeated three times independently with similar results, and f was repeated once. For gel source data, see Supplementary Fig. 1.
a, Extracts from the 293T cells with or without NUFIP1 immunoblotted with the indicated antibodies after 10h of AA withdrawal, showing the same level of mTOR inhibition and r-proteins abundance regardless of the NUFIP1 deletion. Three biologically independent samples are blotted except two samples for NUFIP+/+ -AA condition. b, Volcano plot (−Log10 p-value versus Log2 ratio (NUFIP1−/−/WT)) of 293T cells with or without NUFIP1 deletion (n=7032 proteins). For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 5. n=3 biologically independent samples per genotype. c, Normalized TMR signal in HCT116 RPS3-Halo NUFIP1+/+ or −/− cells incubated with or without 200 nM Torin for 24h, followed by 1h TMR ligand treatment and flow-cytometry analysis. Mean of the triplicate data is plotted. Error bars represent ±SD. d, Average ratio of pre-existing to newly synthesized RPS3-Halo per cell with or without NUFIP1 plotted as a bar graph. Pre-existing Ribo-Halo proteins were labeled with TMR-ligand (100 nM, 1h), followed by the addition of 50 nM Green-ligand. n=3 biologically independent samples. mean ±SD. e, Live-cell imaging of indicated Ribo-Halo cells with or without NUFIP1 labeled with TMR (for pre-existing r-proteins) and Green (for newly synthesized r-proteins) ligands with or without Tor1 (200 nM, 14h). Scale bar = 20 μm. f, Schematic description of the triple TMT-MS analysis of the whole cell lysates gathered from WT 293T or RPS3-Halo 293T cells with or without NUFIP1 after nutrient stress for 10h. g, Lysates of cells treated as in panel F were immunoblotted against the indicated antibodies for quality control, showing that mTOR activity was properly inhibited in all three cell types. h, Volcano plots (−Log10 p-value versus Log2 ratio Nutrient stress/Untreated) of the cells treated as in panel f (WT n=2072 proteins; RPS3-Halo n=2105 proteins; RPS3-Halo and NUFIP1−/− n=2241 proteins). Introducing HaloTag at the endogenous locus did not alter the mTOR inhibition nor ribosome abundance change after nutrient stress. Deletion of NUFIP1 did not show detectable difference either. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 5. n=3 (UT); 4 (-AA, Tor1) biologically independent samples per cell line. i, Immunoblotting of the cell lysates prepared as in panel F shows that introducing HaloTag at the endogenous locus did not alter the mTOR inhibition nor ribosome abundance change after nutrient stress. Deletion of NUFIP1 did not show detectable difference either, consistent with the TMT-MS analysis. j, Keima processing assay using the lysates from HEK293 RPS3-Keima WT or NUFIP−/− cells after the indicated nutrient stress. k, HEK293T WT and NUFIP1−/− cells were left untreated or treated with -AA medium for 3h. Cells were imaged by confocal microscopy after staining with α-NUFIP1 (top) and α-Lamp1 (bottom). Also see Supplementary Table 5. Experiments in e were repeated twice with similar results, and g,i,j were repeated three times independently with similar results. For gel source data, see Supplementary Fig. 1.
a, 293T cells were treated with either Tor1 or 6-MP or were incubated in media lacking Leu, Arg, or AAs for the indicated times and cell extracts subjected to immunoblotting with the indicated antibodies. b, Quantification of the WB data in panel a and c. c, Puromycin incorporation assay after treating 293T cells with the indicated medium and time course. Immunoblotting against anti-puromycin antibody was probed using either infra-red fluorophore labeled 2nd antibody coupled with Odyssey or Horseradish peroxidase labeled 2nd antibody coupled with ECL for comparison. d, Histograms show the quantification of the relative abundance of pre-existing (red) and newly synthesized (green) RPS3-Halo in Fig. 4b. mean, n=2. e, Cell extracted treated as indicated were analyzed for either mTOR inhibition or translatome using AHA incorporation assay coupled with TMR-alkyne click. f, Time-course protein synthesis assay was performed using AHA clicked with TMR-alkyne method after the indicated nutrient stress. g,h, Cells treated as in Fig. 4c were clicked with TMR-alkyne and analyzed by in-gel fluorescence signal (panel g). Immunoblot assays using the indicated antibodies for quality control is shown in panel h (top), and relative TMR signal is plotted below. Centre data are mean ±SD. n=1, 3, 3, 3, 3 and 3 biologically independent samples, from left to right. i, Lysates from HEK293 RPS3-Keima cells after the indicated nutrient stress were immunoblotted against antibodies for Keima or mTOR substrates (top). Abundance ratio of the processed Keima to the intact Keima is plotted (bottom). Related to Fig. 4. Experiments in a,c,g,i were repeated three times independently with similar results, and e,f,i were performed once. For gel source data, see Supplementary Fig. 1.
a, Schematic diagram indicating the points of intersection of nucleotides availability with net ribosome production. 6-MP is an inhibitor of purine biosynthesis and blocks production of rRNAs. b, 293T cells were treated with or without Tor1, 6-MP, or -Arg medium for the indicated time duration. Azidohomoalanine (AHA, 250 μM) was added 3 hours before collecting the cells. Lysates were either immunoblotted against the indicated antibodies for mTOR signaling inhibition (bottom) or processed for TMR-click reaction for in-gel fluorescence analysis (top). c, 293T cells treated with 6-MP for the indicated time points as well as AHA (for the last three hours) were analyzed for either mTOR inhibition (middle) or translatome using AHA incorporation assay coupled with TMR-alkyne click (top). Relative translation efficiency is plotted (bottom). Centre data are mean ±SD. n=1, 3, 3, 3 and 3 biologically independent samples, from left to right. d, 293T cells were incubated in the presence or absence of 6-MP for the indicated times, and AHA (250 μM) was added 3h before collecting each lysate. The translatome was analyzed by biotinylation of AHA-labeled proteins followed by TMT-based proteomics. A volcano plot (−Log10 p-value versus Log2 6-MP/UT) showing the translatome and individual r-proteins (in red) at three time points. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 6. n=1 (Neg Ctrl); n=3 (UT, 9h); 2 (6h, 18h) biologically independent samples per cell line. e, Pre-existing RPS3 in HCT116 RPS3-Halo cells was labeled with TMR (red), washed and then incubated with media with green Halo ligand with or without 6-MP (24h). Live cells were imaged. f, HCT116 RPS3-Halo cells were subjected to 2-color labeling as in Fig. 2f. Cells were either left untreated or incubated with 6-MP prior to analysis of pre-existing and newly synthesized RSP3-Halo using in-gel fluorescence signal-based quantification. Histograms show the relative abundance of pre-existing (red) and newly synthesized (green) RPS3-Halo. Centre data are mean ±SD. n=2, 3, 3, 3, 3, 3 and 3 biologically independent samples, from left to right for both histograms. g, Total proteome analysis of 293T cells (with or without ATG5) was performed according to the scheme (top). Volcano plots (−Log10 p-value versus Log2 6-MP/UT) for all quantified proteins (n=8234 proteins), including individual r-proteins marked with a red dot, are shown at the bottom. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 6. n=3 (UT); 2 (6-MP) biologically independent samples per cell line. h, Keima processing assay using the lysates from HEK293 RPS3-Keima WT or ATG5−/− cells after 6-MP treatment (24h). Cells treated with arsenite was also blotted as a positive control, as it was previously reported to induce selective ribophagy. i,j, Analysis of ribosome concentration using biosynthetic, degradative and cell division information is shown as a simple equation in panel i. Panel j provides a change of ribosome concentration in response to nutrient stress using 0.2 as the degradation rate (derived from AHA-degradomics measurements), translation rates (Tr) of 0.35 derived from AHA-translatome analysis, and a cell cycle factor of 1.5 (Y=1+t/24, t=12h) derived from the proliferation assay. We find that the ribosome concentration upon 12h of Tor1 treatment [0.944*A0/V0} is comparable to the reduction in ribosomes we measured by total proteome analysis (reduction of ribosomes from ~5–8%). Summary of the systematic quantitative analysis of ribosome inventory during nutrient stress. Related to Fig. 4. Experiments in b were performed once, and e,h were repeated three times independently with similar results. For gel source data, see Supplementary Fig. 1.
a, Factors affecting net balance of ribosomes upon nutrient stress. b, 11-plex TMT pipeline to measure ribosome abundance upon nutrient stress +/− active autophagy. Normalized total cell extracts were processed for 8 11-plex TMT-MS3 experiments. c-e, Volcano plots (−Log10 p-value versus Log2 ratio of -AA/UT) for 293T cells (n=8029 proteins), ATG7−/− (n=8373 proteins) or RB1CC1−/− (n=8332 proteins) (panel c), 293 cells (n=7531 proteins), ATG5−/− (n=7504 proteins) (panel d), or HCT116 cells (n=3779 proteins), ATG5−/− (n=3761 proteins) or RB1CC1−/− (n=3671 proteins) (panel e). n=3 (WT); 4 (-AA) biologically independent samples (c-e). For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p-values and q-values, see Supplementary Table 1. f, Mean ratio value measured for ≥ 70 r-proteins treated as in panel b (n=72, 74, 71, 72, 70, 70, 70 and 70 r-proteins, from left to right). Error bars represent SD for n=3 (WT) or n=4 (-AA and Tor1) biologically independent samples. 293T data from. g, Plots of relative abundance of individual r-protein in 293T cells upon either 10h of AA withdrawal (left panel) or Tor1 treatment (right panel). 48 r-proteins with less than ±10% error range for every condition are plotted. Centre data are mean ±SD for n=3 (WT) or n=4 (-AA and Tor1) biologically independent samples. See also Extended Data Fig. 1 and Supplementary Table 1.
a, Contribution of dilution by cell division to net ribosome balance upon nutrient stress. b, Measurement of r-protein concentration in single cells using one-color Halo labeling. c, Normalized TMR signal in RPS3-Halo (>3×105) and RPL29-Halo (>4×103) cells incubated +/− 200 nM Tor1 (14h), followed by 1h TMR ligand treatment and flow-cytometry. Representative of 3 independent experiments. d, Mean (± SD) of triplicate data from panel c is plotted. e, Pulse-chase Ribo-Halo in HCT116 cells. Pre-existing RPS3-Halo was labeled with TMR-ligand (1h) and newly synthesized RPS3-Halo was labeled with green R110-ligand for 8, 16 or 24h (top panel) prior to flow cytometry (frequency histogram, lower left panel, ~1.4×104 cells analyzed). Average from the triplicate experiments (bottom right panel). Error bars; SD. See METHODS for details. f, Scheme for two-color Ribo-Halo r-protein biogenesis and dilution labeling +/− Tor1 (top panel). Pre-existing RPS3-Halo was labeled with TMR-ligand (1h), and newly synthesized RPS3-Halo +/− Tor1 (200 nM) was labeled with green R110-ligand. The ratio of 561/620 to 488/550 plotted against cell populations (bottom left panel), and the mean values from triplicate experiments of 8h, 16h, and 24h pulse chase (bottom right panel) are shown. Error bars; SD. g, Imaging of pre-existing TMR-labeled RPS3-Halo and newly synthesized green R110-ligand labeled RPS3-Halo Green +/− Tor1 (200 nM, 14h). Scale bar = 20 μm. h, In-gel fluorescence of RPS3-Halo as in panel f. Gels were then transferred for immunoblotting with α-RPS3. Experiments shown in g-h were repeated >3 times with similar results. Full immunoblot is shown in Extended Data Fig. 11f. See also Extended Data Fig. 2, 3. For gel source data, see Supplementary Fig. 1.
a, Contribution of r-protein synthesis to net ribosome balance upon nutrient stress. b, AHA-based translatomics to measure translational suppression during nutrient stress. c, Extracts from 293T cells +/− AA or Tor1 treatment in the presence of Met or AHA (250 μM, 3h each) were labeled with TMR and analyzed by in-gel fluorescence. n=3 biologically independent samples for UT, -AA, and Tor1, and n=1 for Met. d, 293T extracts (panel b) were clicked with biotin prior to streptavidin enrichment and proteomics. A plot of −Log10 p-value versus Log2 ratio of Tor1/untreated for quantification (n=8285 proteins) is shown. n=3 biologically independent samples. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p- and q-values, see Supplementary Table 2. e, Violin plots for TMT intensity of newly synthesized proteins in WT, ATG7−/−, or RB1CC1−/− 293T cells (n=8285, 3336, 3418 proteins, respectively). Black horizontal lines: median abundance of r-proteins (black circles). Colored horizontal dotted lines: median of all proteins. Violin plots represent the distribution and density of the whole dataset (Center-line: median; Limits: minima and maxima). f, Contribution of r-protein degradation to net ribosome balance upon nutrient stress. g, Schematic of degradomics analysis using AHA pulse labeling (5h) to examine proteome turnover and autophagy dependence during mTOR inhibition (12h). Extracts were immunoblotted or clicked with biotin for TMT-proteomics. h, Pattern 1: accelerated degradation via autophagy. Pattern 2: accelerated degradation independent of autophagy. Pattern 3: stabilization independent of autophagy. n=10 proteins’ average values from biological triplicate experiments; mean±SEM. i, Volcano plots (−Log10 p-value versus Log2 Tor1/UT 12h) for 293T WT, ATG7−/−, and RB1CC1−/− cells (n=8304, 8319, 8590 proteins, respectively). r-proteins, red. n=3 biologically independent samples. For two-sided Welch’s t-test (adjusted for multiple comparison) parameters, individual p- and q-values, see Supplementary Table 3. j, Average ratio for AHA-labeled r-proteins as in panel g. n=58 r-proteins (STDEV < 0.3 filter for every condition, n=3 biologically independent samples). mean±SEM. See also Extended Data Fig. 4–6 and Supplementary Table 2,3. For gel source data, see Supplementary Fig. 1.
a, Points of intersection of individual AAs with net ribosome production. b, HCT116 RPS3-Halo cells were either left untreated, subjected to AAs, Leu, or Arg withdrawal or incubated with Tor1 prior to analysis of pre-existing and newly synthesized RPS3-Halo as in Fig. 2f. For quantification of n=2 biologically independent experiments, see Extended Data Fig. 10d. c, Experimental workflow for AHA translatome analysis of 293T cells with or without the indicated AAs or with Tor1. d, Violin plots displaying the relative proteome translation rates and the effect of removal of indicated AAs or mTOR inhibition on the translation of individual r-proteins (black circles). The median abundance of r-proteins is indicated by the black horizontal lines and the median of all proteins is indicated by the colored horizontal dotted lines. The violin curves represent the distribution and density of the whole dataset (Center-line: median; Limits: minima and maxima). e, Ribosome homeostasis framework. Left panel: points of regulation, with blue arrows indicating the net effect of nutrient stress. Right panel compares the effect of nutrient stress on global proteome and r-protein translation and degradation, primarily through non-autophagic mechanisms, and the effect of nutrient stress on r-protein dilution via cell division. Ribosome concentration upon nutrient stress in a population of cells will reflect a decrease in r-protein number through translational and degradative mechanisms corrected for by changes in cell volume and cell division. See also Extended Data Fig. 10 and Supplementary Table 6. For gel source data, see Supplementary Fig. 1.
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