Proteome Turnover in the Green Alga Ostreococcus tauri by Time Course ¹⁵N Metabolic Labeling Mass Spectrometry

Abstract

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

“…Another approach is to supply 15 NO 3 or 15 NH 4 to the medium of algae (Martin et al, 2012;Mastrobuoni et al, 2012) or the rooting medium of higher plants (Masclaux-Daubresse and Chardon, 2011;Nelson et al, 2014aNelson et al, , 2014b. Labeling with 15 N is especially useful in studies that use liquid chromatographytandem mass spectrometry to analyze the labeling kinetics of signature peptides of individual proteins Nelson et al, 2014aNelson et al, , 2014b.…”

Clocks in Algae

“…The lack of a cellulose cell wall facilitates simple organelle enrichment and protein extraction, allowing large-scale analysis of the O. tauri proteome and phosphoproteome by massspectrometry. In O. tauri grown 12 h/12 h LD cycles, 27% of the predicted proteome was detected (97,98) , including phosphopeptides from proteins relevant to the circadian clock (CO, CRY1, LHY, TOC1, CK1 and GSK3). No forward genetic methods have been developed as yet, hampering the identification of clock 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 components that lack homology to those of other organisms, but a gene knock-out protocol using homologous recombination was recently published (95) .…”

The reduced kinome of Ostreococcus tauri: core eukaryotic signalling components in a tractable model species

“…It has already been used as a model for the eukaryotic cell-cycle, helping to unify current understanding of cell-cycle regulation across eukaryotes [23]. The lack of a cellulose plant cell wall facilitates transformation [24, 25] as well as organelle enrichment and protein extraction [26, 27]. These genetic and proteomic tools have already been applied to studies of protein turnover [27], nutrient deprivation [26] and the plant circadian clock in experimental [25, 28, 29] and mathematical approaches [30].…”