The composition and turnover of the Arabidopsis thaliana 80S cytosolic ribosome

Abstract

Cytosolic 80S ribosomes contain proteins of the mature cytosolic ribosome (r-proteins) as well as proteins with roles in ribosome biogenesis, protein folding or modification. Here, we refined the core r-protein composition in Arabidopsis thaliana by determining the abundance of different proteins during enrichment of ribosomes from cell cultures using peptide mass spectrometry. The turnover rates of 26 40S subunit r-proteins and 29 60S subunit r-proteins were also determined, showing that half of the ribosome population is replaced every 3-4 days. Three enriched proteins showed significantly shorter half-lives; a protein annotated as a ribosomal protein uL10 (RPP0D, At1g25260) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The At1g25260 protein is a homologue of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role in the 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins; 26 were enriched more than 10-fold during ribosome enrichment. Some of these enriched proteins have known roles in translation, while others are newly proposed ribosome-associated factors in plants. This analysis provides an improved understanding of A. thaliana ribosome protein content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and how protein turnover can help identify r-proteins involved in ribosome biogenesis or regulation in plants.

Keywords: Arabidopsis thaliana; proteomics; ribosomes.

Figure 1.. Purification and composition of the 80S cytosolic ribosome in Arabidopsis cell culture.

(A) Number of identified Arabidopsis proteins (AGIs) found in all of three replicates following differential centrifugation steps for low, partial and high enrichment of ribosomes. (B) The number of Arabidopsis 80S r-proteins isoforms identified across the three replicates. (C) Percentage of normalised spectral abundance factors (NSAF) in each ribosome enrichment that are r-proteins. (D) The ratio of NSAF for 60S large subunit r-proteins/40S small subunit r-proteins (RPL/RPS). (E) Principal component analysis of spectral counts assigned to the 319 proteins found by the differential centrifugation process across the three replicates and three purification steps (1–9). (F) Hierarchical clustering analysis of spectral counts for the 319 proteins identified in the purification process. The relative number of normalised spectral counts is shown in green for numbers <1 and in red for numbers >1. The vertical numbers (1–6) indicate cluster number based on Pearson correlation. Cluster 6 contains 193 proteins, including 135 80S cytosolic ribosome r-proteins. The horizontal numbers (1–9) indicate replicate number as in (E).

Figure 2.. Frequency distribution graphs of the ratio of spectral counts for r-proteins and ribosome-associated proteins in pairs of ribosomal enrichments.

(A) Frequency histogram of the ratio of spectral counts in samples of different enrichment for r-proteins and ribosome-associated proteins; the blue bars indicate the distribution of ratios for r-proteins and red bars indicate the distribution of rations for ribosome-associated proteins. (B) Cumulative relative frequency distribution graphs of (A). Statistical significance was estimated by the Kolmogorov–Smirnov test for 193 proteins including 135 r-proteins (blue) and 58 ribosome-associated proteins (red) (P < 0.0001) from cluster 6 (Figure 1E).

Figure 3.. Protein turnover of ribosomes in Arabidopsis cell culture.

(A) Growth rate of Arabidopsis cell culture over 9 days after transferred into new media (FW, n = 4, standard error). (B) Box plot of the average ¹⁵N enrichment of r-protein peptides after 1, 3 and 5 days of growth in 98% ¹⁵N media. The number of r-proteins included for each time point is shown above the box plot (n = 4). (C,D) box plots representing the average degradation rate (K_d) of r-proteins after day 1, 3 and 5 through the course of the growth experiment. (C) K_d for each time point using measured FCP values. (D) K_D using the median polish strategy for FCP calculation.

Figure 4.. Turnover rate of specific r-proteins in Arabidopsis cell culture.

(A) Degradation rate of core 60S ribosomal subunit proteins and the putative 60S-transacting factor Mrt4/RPP0D. (B) Degradation rate of 40S ribosomal subunit proteins. In both (A) and (B) the mean degradation rate value for each protein was calculated using data from ≥ 3 biological replicates and included data from ≥ 5 peptide spectra. Error bars: standard error. * indicates significant difference from the mean of the ribosome (based on t-tests P < 0.05). (C) Box plots of K_D values for r-proteins of the 60S large and 40S small subunits.