Investigating the effect of Target of Rapamycin kinase inhibition on theChlamydomonas reinhardtiiphosphoproteome: from known homologs to new targets

Abstract

1• Target of Rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is 2 modulated in response to nutrients, energy and stress. Key proteins involved in the pathway 3 are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the 4 substrates of TOR kinase and downstream signaling network have not been elucidated. Our 5 study provides a new resource for investigating the phosphorylation networks governed by the 6 TOR kinase pathway in Chlamydomonas. 7• We used quantitative phosphoproteomics to investigate the effects of inhibiting 8 Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-9 insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors 10 (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were 11 identified. 12 • Our quantitative phosphoproteomic dataset comprised 2,547 unique phosphosites from 1,432 13 different proteins. Inhibition of TOR kinase caused significant quantitative changes in 14 phosphorylation at 258 phosphosites, from 219 unique phosphopeptides. 15 • Our results include Chlamydomonas homologs of TOR signaling-related proteins, including a 16 site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins 17 involved in translation and carotenoid biosynthesis were identified. Follow-up experiments 18guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that 19 carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR 20 control in algae. 21

Reciprocal regulation of photosynthesis and mitochondrial respiration by TOR kinase in Chlamydomonas reinhardtii

“…NPQ involves the dissipation of excess energy by the photosynthetic apparatus, activated during high light acclimation, change in CO 2 and stress levels via three major contributing components, namely, qE (involving pigments such as xanthophylls and other carotenoids), qT (involving state transitions), and qI (D1 core damage repair) (Erickson et al, ; Müller, Li, & Niyogi, ). Recently, it has been shown that carotenoid production is under the control of TOR kinase, thus rationalizing its role in the regulation of qE (Werth et al, ). Moreover, our study shows increase in the levels of LHCSR3 upon TOR kinase inhibition in comparison to untreated cells, which suggests that the qE component of NPQ is active (Figure e; Figure c).…”