Global proteome turnover analyses of the Yeasts S. cerevisiae and S. pombe

Abstract

How cells maintain specific levels of each protein and whether that control is evolutionarily conserved are key questions. Here, we report proteome-wide steady-state protein turnover rate measurements for the evolutionarily distant but ecologically similar yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We find that the half-life of most proteins is much longer than currently thought and determined to a large degree by protein synthesis and dilution due to cell division. However, we detect a significant subset of proteins (∼15%) in both yeasts that are turned over rapidly. In addition, the relative abundances of orthologous proteins between the two yeasts are highly conserved across the 400 million years of evolution. In contrast, their respective turnover rates differ considerably. Our data provide a high-confidence resource for studying protein degradation in common yeast model systems.

Fig. 1. Quantifying protein turnover in S. cerevisiae and S. pombe

(A) Experimental design for turnover measurements in yeasts. Decay curves for the indicated proteins in S. cerevisiae and S. pombe are shown. (B) Mass spectra after labeling of a peptide from Eno2 (AADALLK2+) in S. cerevisiae. The old peptide (“heavy”, red) decays as the newly synthesized peptide (“light”, green) increases in intensity during the time course of the experiment. (C) Log₂(H/L) intensities from 40,452 peptides measured in two independent biological replicates. The insert shows the histogram distribution of log₂(H/L) (mean = −0.0018, SD = 1.26 expressed as a fold-change from the mean). (D) Histograms of protein half-lives in S. cerevisiae (red, dashed line indicates the median half-life = 8.8 hours) and S. pombe (blue, dashed line indicates the median half-life = 11.1 hours).

Fig. 2. Protein half-life differences in distinct sets of proteins

(A) Contributions of degradation (K_deg) and protein dilution due to cell growth (K_dil) in S. cerevisiae and S. pombe. Log₂(K_deg/K_dil) ratios define three classes of protein abundance regulation: class I (K_deg ≥ 2xK_dil), class II (0.5xK_dil ≤ K_deg ≥ 2xK_dil) and class III (K_deg ≤ 2xK_dil). (B) Gene Ontology (GO) analysis of class I and class II proteins in S. cerevisiae. (C) Comparison of protein abundances with ribosome footprint data in S. cerevisiae for proteins of class I (purple), class II (orange) and class III (green). (D) Comparison of protein abundances between class I, II and III. (E) Sequence analysis of the 5′UTR (30-mer ahead of start codon) of the 10% most abundant proteins in S. cerevisiae.

Fig. 3. Protein abundances, but not half-lives are evolutionarily conserved between S. cerevisiae and S. pombe

(A) Scatter plot comparing homologous protein abundances. (B) Scatter plot comparing homologous protein half-lives. (C) Cumulative density distribution of protein half-lives. (D) Half-live comparison of the large (RLP, MRLP) and the small (RSP, MRSP) subunits of the cytosolic and mitochondrial ribosomes. (E) Half-live comparison of proteins involved in the arginine biosynthetic pathway.

Fig. 4. Quantitative turnover analysis reveals the evolution of different strategies to control ergosterol metabolism enzymes

(A) The abundance of ergosterol synthetic enzymes is conserved in S. cerevisiae and S. pombe (yellow). (B) The half-lives of ergosterol synthesis enzymes are similar in S. pombe whereas in S. cerevisiae, Erg1, Erg11, Erg3, Erg25 and Erg5 are short-lived proteins. In blue are anaerobically induced and Sre1 dependend gene in S. pombe and their counterpart in S. cerevisiae. (C) Plot of half-lives (yellow), transcripts abundances (light grey) and ribosome footprint (dark grey) for ergosterol synthesis enzymes. (D) Erg1 and Erg25 degradation followed by SILAC labeling decay in wild-type and hrd1Δ strains. (E) Half-live (yellow) and abundance (grey) fold changes of the indicated proteins in hrd1Δ compared with wild-type strains. (F) Representation of the regulation of the S. cerevisiae's short-lived ergosterol metabolic enzymes (green, left) and their orthologs in S. pombe (right). ERAD ubiquitin ligases are in red and previously characterized regulations are indicated in dashed dark red and new in dashed bright red. Previously described transcription factors regulation is indicated in blue.