Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii

Abstract

Rapamycin is an inhibitor of the evolutionary conserved Target of Rapamycin (TOR) kinase which promotes and coordinates translation with cell growth and division. In heterotrophic organisms, TOR regulation is based on intra- and extracellular stimuli such as amino acids level and insulin perception. However, how plant TOR pathways have evolved to integrate plastid endosymbiosis is a remaining question. Despite the close association of the TOR signaling with the coordination between protein turn-over and growth, proteome and phosphoproteome acclimation to a rapamycin treatment have not yet been thoroughly investigated in <i>Chlamydomonas reinhardtii</i>. In this study, we have used <i>in vivo</i> label-free phospho-proteomic analysis to profile both protein and phosphorylation changes at 0, 24, and 48 h in <i>Chlamydomonas</i> cells treated with rapamycin. Using multivariate statistics we highlight the impact of TOR inhibition on both the proteome and the phosphoproteome. Two-way ANOVA distinguished differential levels of proteins and phosphoproteins in response either to culture duration and rapamycin treatment or combined effects. Finally, protein-protein interaction networks and functional enrichment analysis underlined the relation between plastid and mitochondrial metabolism. Prominent changes of proteins involved in sulfur, cysteine, and methionine as well as nucleotide metabolism on the one hand, and changes in the TCA cycle on the other highlight the interplay of chloroplast and mitochondria metabolism. Furthermore, TOR inhibition revealed changes in the endomembrane trafficking system. Phosphoproteomics data, on the other hand, highlighted specific differentially regulated phosphorylation sites for calcium-regulated protein kinases as well as ATG7, S6K, and PP2C. To conclude we provide a first combined <i>Chlamydomonas</i> proteomics and phosphoproteomics dataset in response to TOR inhibition, which will support further investigations.