Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes

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.

Photosynthetic Metabolism and Nitrogen Reshuffling Are Regulated by Reversible Cysteine Thiol Oxidation Following Nitrogen Deprivation in Chlamydomonas

“…Chloroplastic ω-3-fatty acid desaturase (Cre01.g038600.t1.2) and chloroplastic ω-6-fatty acid desaturase (Cre13.g590500.t1.1) each showed significantly decreased cysteine oxidation, the former showing a FC l2 of −1.6 on C75 and C230 and the latter showing a FC l2 of −1.4 on C387. Our previous work associated an increase of reversible oxidation on C387 with the inhibition of target of rapamycin (TOR), a master-regulatory kinase whose inhibition causes phenotypic changes similar to those seen under nitrogen deprivation [ 33 ]. Similarly, our previous study revealed a 2.2 FC l2 on C653 of the phospholipase B-like protein (Cre03.g182750.t.2), whereas this current study showed a FC l2 of 3.3 on the same cysteine.…”

“…Reversible thiol oxidation was enriched using an oxidized cysteine resin-assisted capture OxRAC) strategy, as described previously ( Figure 1 ) [ 21 , 33 ]. Briefly, 1 mg of protein lysates were incubated with 10 mM DTT on a Thermomixer in constant darkness at 25 °C and 1000 rpm for 1 h in order to reduce all reversibly oxidized cysteines.…”

“…Data were parsed using custom scripts written in R programming for pre-processing and statistical analysis, as previously described [ 33 ]. To test for significantly changing oxidation and global abundance between the nitrogen deprived and replete conditions, a two-sided, equal variance t -test was performed.…”

“…While irreversible oxidation is associated with large scale protein damage, reversible oxidation, particularly of cysteine thiols, acts as a protein switch similar to that of phosphorylation, in which temporary oxidation may modulate protein activity [ 32 ]. Reversible oxidation in the form of disulfide bonds, S -glutathionylation, S -nitrosylation, and S -sulfonation has been implicated in the regulation of signaling, stress response, transcription, translation, and carbon metabolism, as well as photosynthesis [ 33 , 34 , 35 , 36 , 37 ]. While nitrosylation, glutathionylation, and the thioredoxome have been explored in C. reinhardtii , the extent to which oxidative signaling is used to respond and acclimate to nitrogen deprivation is poorly understood [ 26 , 27 , 28 ].…”

Contributions of TOR Signaling on Photosynthesis

“…Researchers examined changes in oxidation during TOR inhibition in green alga Chlamydomonas reinhardtii. A reduction in photosynthetic electron flow and twenty proteins related with photosynthesis were identified with altered oxidation status under TOR inhibition [66].…”