Abstract
The target of rapamycin (TOR) kinase is a master metabolic regulator with roles in nutritional sensing, protein translation, and autophagy. In Chlamydomonas reinhardtii, a unicellular green alga, TOR has been linked to the regulation of increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target(s) is responsible for the elusive “lipid switch” in control of increasing TAG accumulation under nutrient limitation. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems, and little work has been done to show the role of oxidative signaling in TOR regulation. In this study, the TOR inhibitor AZD8055 was used to relate reversible thiol oxidation to the physiological changes seen under TOR inhibition, including increased TAG content. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 401 proteins were determined to have significant changes in oxidation following TOR inhibition. These oxidative changes mirrored characterized physiological modifications, supporting the role of reversible thiol oxidation in TOR regulation of TAG production, protein translation, carbohydrate catabolism, and photosynthesis through the use of reversible thiol oxidation. The delineation of redox-controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.
Highlights
target of rapamycin (TOR) is involved in the regulation of protein synthesis, with the small molecule inhibitor rapamycin decreasing protein synthesis through the phosphorylation of BiP, an endoplasmic reticulum chaperone and member of the HSP70 superfamily involved in post-translational protein folding [27]
TOR has a significant role in the overall nutrient metabolism of the cell, including the aforementioned TAG synthesis pathways as well as the tricarboxylic acid cycle, which is downregulated under TOR inhibition and decreases carbohydrate catabolism [18,28]
Proteins with significantchanges changes in oxidation, focusing on the proteins involved in lipid synthesis, protein translation, carbohydrate focusing on the proteins involved in lipid synthesis, protein translation, carbohydrate metabolism, thepathway, TOR pathway, and photosynthesis, it was to possible to observed integrate physiological observed metabolism, the TOR
Summary
Target of rapamycin (TOR) is a conserved Ser/Thr kinase and master regulator of cellular growth and homeostasis in eukaryotes, with significant control over nutrient-responsive pathways including macromolecular anabolism and catabolism as well as vacuole formation and autophagy [1,2,3,4,5,6,7].In yeast, mammals, and other complex eukaryotes, TOR has been identified in two distinct complexes, TORC1 and TORC2 [2,8,9,10,11], but in photosynthetic organisms only the components of TORC1 have been identified [2,4]. TOR results in similar phenotypic changes observed under nitrogen deprivation [19,20], including an increase in triacylglycerol (TAG) content and a decrease in protein synthesis [21]. This suggests that the eukaryotic “lipid switch”—the protein(s) responsible for upregulating lipid formation under nutrient limitation—is regulated by the TOR pathway [19,20,21,22,23]. TOR has a significant role in the overall nutrient metabolism of the cell, including the aforementioned TAG synthesis pathways as well as the tricarboxylic acid cycle, which is downregulated under TOR inhibition and decreases carbohydrate catabolism [18,28]
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