Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation

Abstract

The Target of Rapamycin (TOR) kinase is a central regulator of growth and metabolism in all eukaryotic organisms, including animals, fungi, and plants. Even though the inputs and outputs of TOR signaling are well characterized for animals and fungi, our understanding of the upstream regulators of TOR and its downstream targets is still fragmentary in photosynthetic organisms. In this study, we employed the rapamycin-sensitive green alga to elucidate the molecular cause of the amino acid accumulation that occurs after rapamycin-induced inhibition of TOR. Using different growth conditions and stableC- and N-isotope labeling, we show that this phenotype is accompanied by increased nitrogen (N) uptake, which is induced within minutes of TOR inhibition. Interestingly, this increased N influx is accompanied by increased activities of glutamine synthetase and glutamine oxoglutarate aminotransferase, the main N-assimilating enzymes, which are responsible for the rise in levels of several amino acids, which occurs within a few minutes. Accordingly, we conclude that even though translation initiation and autophagy have been reported to be the main downstream targets of TOR, the upregulation of de novo amino acid synthesis seems to be one of the earliest responses induced after the inhibition of TOR in Chlamydomonas.

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

“…As discussed above, the role of TOR kinase has also been extensively studied using two inhibitors rapamycin and AZD8055 in the unicellular eukaryotic algae, Chlamydomonas reinhardtii. As expected, TOR kinase plays a central role in growth, metabolism, ER stress, autophagy, carbon/nitrogen balance, and lipid accumulation in C. reinhardtii (Juppner et al, 2018;Mubeen et al, 2018;Perez-Perez, Couso, & Crespo, 2017). The role of TOR kinase in cellular phosphate metabolism was also uncovered in TOR hypersensitive mutants revealing lower levels of InsP 6 and InsP 7 (inositol phosphates) (Couso et al, 2016).…”

“…The role of TOR kinase in cellular phosphate metabolism was also uncovered in TOR hypersensitive mutants revealing lower levels of InsP 6 and InsP 7 (inositol phosphates) (Couso et al, ). Counterintuitively, one of the recent studies concludes that TOR kinase inhibition positively regulates nitrogen assimilation leading to increased ammonium uptake and amino acid biosynthesis (Mubeen et al, ). Interestingly, a recent study in C. reinhardtii based on proteomics analysis suggested an overall reduction in the proteome of mitochondrial TCA cycle following TOR kinase inhibition (Roustan & Weckwerth, ).…”

“…Moreover, mammalian TORC1 can directly sense nutrients such as amino acids via the activation of RAG GTPases that recruit TOR kinase to the lysosomal membrane, whereas Arabidopsis and green algae lack these RAG like GTPases and other amino acid sensors (van Dam, Zwartkruis, Bos, & Snel, 2011;Gonzalez & Hall, 2017; Kim, Goraksha-Hicks, Li, Neufeld, & Guan, 2008;Shi et al, 2018;Xiong & Sheen, 2014). Interestingly, other nutrients such as nitrogen (N), phosphate (Pi), and sulfur (S) that play an important role in plant growth and development also act via the TOR kinase pathway, thus indicating unique plant-associated sensing components that could directly or indirectly activate TOR kinase in many plant species including rice and Arabidopsis (Bakshi, Moin, Datla, & Kirti, 2017;Bakshi, Moin, Madhav, & Kirti, 2019;Couso et al, 2016;Dong et al, 2017;Mubeen, Jüppner, Alpers, Hincha, & Giavalisco, 2018;Shi et al, 2018;Speiser et al, 2018;Wu et al, 2019;Xiong & Sheen, 2015). These studies suggest that plants may have evolved unique components to regulate growth and metabolism and the detailed characterization of the same in plants is still under progress.…”

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of Rapamycin (TOR) Kinase Activation

“…In this regard, the general upregulation of amino acid catabolic genes, including PDH1, by inactivation of TOR has been repeatedly observed in previous plant transcriptome analyses (Ren et al, 2012;Xiong et al, 2013;De Vleesschauwer et al, 2018). In addition, disruption of TOR signaling in plants leads to the accumulation of high levels of amino acids and TCA cycle organic acids (Moreau et al, 2012;Ren et al, 2012;Caldana et al, 2013), potentially due to activation of amino acid synthesis (Mubeen et al, 2018). Our findings provide evidence that TOR activity in plants is influenced by amino acid levels leading to downstream effects on respiratory substrate use, including that of amino acids.…”

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

“…Inhibiting the action of mTORc1, even transiently, is known to lead to an increase in intracellular amino acid concentrations due to decreased sequestering of amino acids in nascently synthesized proteins (Mubeen et al 2018). Thus, it is permissible that the inhibitory effect of rapamycin on 2-deoxyglucose uptake is the result of increased intracellular leucine concentrations as opposed to impaired mTORc1 function per se.…”