Riboproteome remodeling during quiescence exit in Saccharomyces cerevisiae

Abstract

The quiescent state is the prevalent mode of cellular life in most cells. Saccharomyces cerevisiae is a useful model for studying the molecular basis of the cell cycle, quiescence, and aging. Previous studies indicate that heterogeneous ribosomes show a specialized translation function to adjust the cellular proteome upon a specific stimulus. Using nano LC-MS/MS, we identified 69 of the 79 ribosomal proteins (RPs) that constitute the eukaryotic 80S ribosome during quiescence. Our study shows that the riboproteome is composed of 444 accessory proteins comprising cellular functions such as translation, protein folding, amino acid and glucose metabolism, cellular responses to oxidative stress, and protein degradation. Furthermore, the stoichiometry of both RPs and accessory proteins on ribosome particles is different depending on growth conditions and among monosome and polysome fractions. Deficiency of different RPs resulted in defects of translational capacity, suggesting that ribosome composition can result in changes in translational activity during quiescence.

Keywords: Cell biology.

Graphical abstract

Figure 1

Global translational state of quiescent cells before and after stimulation with nutrients (A) A_254 nm traces show the polysome profiles of cells in exponential phase (EP), in stationary phase (SP) or after stimulation with fresh medium for 30 min (SP + 30 min SDC), 60 min (SP + 60 min SDC) and 150 min (SP + 150 min SDC). The 40S, 60S, 80S particles, and polysomes are indicated. Equal amounts of total RNA were used in each gradient. The graph shows the 80S (M)/polysome (P) area, mean ± SE n = 3 biological replicates. Statistical significance was determined with a one-way ANOVA test and Tukey contrasts test (∗p < 0.001; ∗∗∗∗p < 0.0001). n = 3 biological replicates. (B) mRNA levels and (C) distribution in polysomal profiles during quiescence and after fresh medium stimulation. Total mRNAs were measured by Northern blot from cells in the stationary phase (SP) and after 30 min (SP + 30 min SDC) or 60 min (SP + 60 min SDC) of stimulation with fresh medium. 18S rRNA was used as a loading control. mRNA distribution in polysome profile was performed in 15–50% sucrose gradient fractions in SP, SP + 30 min SDC, or SP + 60 min SDC. Samples were pooled depending on the ribosomal content: free are fractions with densities less than 40S (F); monosomal are fractions between 40S and 80S (M), polysomal are fractions over 80S (P); as indicated in the example polysomal profile on the left. The percentage distribution of ENO2, PDC1, PAB1 and NCE102 mRNAs in each fraction measured by RT-qPCR was plotted. Luciferase mRNA was used as a control. The values represent the mean ± SE, n = 2 biological replicates. (D) Protein levels of ENO2-TAP, PDC1-TAP, Pab1, and NCE102-TAP during exponential growth (EP), stationary phase (SP), and after 30 min or 60 min of stimulation with fresh medium (SP + 30 min SDC or SP + 60 min SDC) were determined by Western blot using anti-TAP or anti-Pab1p antibodies. Ponceau red was used as a loading control. Quantification of Western blot was performed by densitometry (mean ± SE, n = 3 biological replicates). Statistical significance for each protein was determined with a one-way ANOVA and the Tukey contrast test ∗∗∗∗p < 0.0001.

Figure 2

Riboproteome characterization during quiescence and translational recovery (A) Experimental design of the proteomic screen. Stationary phase (SP) cells were harvested by centrifugation and resuspended in the same volume of fresh medium for 30 min (SP + 30 min SDC), or 60 min (SP + 60 min SDC). Free (F), monosomal (M, 40S + 60S + 80S), and polysomal (P) fractions were obtained by ultracentrifugation through sucrose gradients and analyzed by nanoLC-MS/MS (for details, see STAR Methods). (B) Distribution of 527 total proteins detected in all 3 growth conditions through mass spectrometry. The number of proteins for each subgroup is indicated. See also Figure S3.

Figure 3

Analysis of the composition of core ribosomal proteins The relative abundance distribution of the ribosomal proteins of the large and small subunit of the ribosome in monosomal (M) or polysomal (P) fractions. The distribution of different ribosomal proteins in monosomal (M), or polysomal fractions (P) is shown. The emPAI data obtained by nanoLC-MS/MS from stationary phase (SP) or after 60 min of fresh medium stimulus (SP + SDC 60 min) riboproteome were used to calculate the relative protein abundance distribution. Heat maps indicate the distribution of relative abundance between the fractions M and P in each condition. The color scale is indicated, ND = not detected. Relative abundance distribution less than 0.8 was not considered. Ribosomal protein paralogs with 100% identity (violet) and those for which it was not possible to distinguish between paralog pairs (green).

Figure 4

Characterization of co-fractionated proteins with the core ribosomal proteins (A) The pie chart shows the number of unique proteins identified in this study, classified by Gene Ontology. (B and C) The relative abundance distribution of different proteins in free (F), monosomal (M), or polysomal fractions (P) is shown. The emPAI data obtained by nanoLC-MS/MS from stationary phase (SP) or after 60 min of fresh medium stimulus (SP + SDC 60 min) riboproteome were used to calculate the relative protein abundance distribution. Heat maps indicate the distribution of relative abundance between the fractions F, M, and P in each condition. The color scale is indicated, ND = not detected. Relative abundance distribution less than 0.8 was not considered. Representative proteins from proteasome, protein folding, translation, and miscellaneous groups are shown (Table S1, Protein abundance F, M, P). Representative proteins of each category are shown, nucleotide biosynthetic process purine (violet), amino acid biosynthesis (pink), mRNA binding (red), oxidative stress (green), cellular transport and large membranous structures (light blue), and mitochondria (brown).

Figure 5

Deletion of specific RPs alters translational activity during quiescent exit (A) Maximum cell density measured at A₆₀₀nm after 72 h growth (n = 6 biological replicates) and cell viability of quiescent cells evaluated by spot assay and phloxine B incorporation. Spotted plates were photographed after 24 h and 48 h (Figure S9A). (B) Cell growth of stationary phase cells stimulated with fresh SDC media (n = 3 biological replicates). The inset shows the lag phase length. Logistic population growth was adjusted using Graphpad Prism 8. (C) Global translational activity was measured by puromycin incorporation during quiescence exit. Neosynthesized proteins were labeled with puromycin and analyzed by Western blot and quantified by densitometry (∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0,05, n = 3 biological replicates, Figure S10). (D) Translation activity was evaluated with Renilla luciferase reporter (n = 3 biological replicates). For the time course data, a two-way ANOVA with Bonferroni’s multiple comparison test was used to assess differences between time points (∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0,05). Control experiments shows that the Renilla mRNA level does not differ between strains (Figure S7B).