Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets

Abstract

Target of rapamycin (TOR) kinase is a conserved regulator of cell growth whose activity is modulated in response to nutrients, energy and stress. Key proteins involved in the pathway are conserved in the model photosynthetic microalga Chlamydomonas reinhardtii, but the substrates of TOR kinase and downstream signaling network have not been elucidated. Our study provides a new resource for investigating the phosphorylation networks governed by the TOR kinase pathway in Chlamydomonas. We used quantitative phosphoproteomics to investigate the effects of inhibiting Chlamydomonas TOR kinase on dynamic protein phosphorylation. Wild-type and AZD-insensitive Chlamydomonas strains were treated with TOR-specific chemical inhibitors (rapamycin, AZD8055 and Torin1), after which differentially affected phosphosites were identified. Our quantitative phosphoproteomic dataset comprised 2547 unique phosphosites from 1432 different proteins. Inhibition of TOR kinase caused significant quantitative changes in phosphorylation at 258 phosphosites, from 219 unique phosphopeptides. Our results include Chlamydomonas homologs of TOR signaling-related proteins, including a site on RPS6 with a decrease in phosphorylation. Additionally, phosphosites on proteins involved in translation and carotenoid biosynthesis were identified. Follow-up experiments guided by these phosphoproteomic findings in lycopene beta/epsilon cyclase showed that carotenoid levels are affected by TORC1 inhibition and carotenoid production is under TOR control in algae.

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, 2015;Müller, Li, & Niyogi, 2001). 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, 2019). 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 2e; Figure S2c).…”

Phosphorus Availability Regulates TORC1 Signaling via LST8 in Chlamydomonas

“…To further characterize the phenotype of lst8-1 cells, we developed a method to monitor TOR kinase activity. Because previous efforts to detect TORC1 activity in Chlamydomonas with commercial anti-phosphoS6K antibodies have failed (Couso et al 2016;Werth et al, 2019), we instead generated antibodies against Chlamydomonas RPS6, a well-defined target of S6K whose phosphorylation is controlled by TORC1 signaling in different organisms, including plants (Dobrenel et al, 2016b). An amino acid alignment of the C terminus of Chlamydomonas, Arabidopsis, yeast (Saccharomyces cerevisiae), and human RPS6 proteins revealed that Ser-245 from Chlamydomonas is highly conserved in other RPS6 proteins (Figure 2A).…”

“…A recent study in Chlamydomonas used quantitative phosphoproteomics following TORC1 inhibition to identify a number of proteins with altered phosphorylation that are involved in translation, carotenoid biosynthesis, or autophagy (Roustan and Weckwerth, 2018;Werth et al, 2019). Metabolomic and transcriptomic studies of Chlamydomonas cells treated with rapamycin showed an important role of TOR in the regulation of primary metabolism, particularly in the de novo synthesis of amino acids (Jüppner et al, 2018;Mubeen et al, 2018).…”

Targeting TOR signaling for enhanced lipid productivity in algae

“…S6Ks are conserved TOR targets found in plants and algae that are known to regulate translation [47,48]. Phosphoproteomic studies of Chlamydomonas cells treated with rapamycin or AZD-8055 identified TOR-dependent phosphorylation sites in S6K and ribosomal S6 protein [43,49]. In mammals, S6K regulates SREBP processing and other lipogenic genes.…”