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Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes
Author(s) -
Smythers Amanda L.,
Ford Megan L.,
McConnell Evan,
Lowery Sarah,
Kolling Derrick R.J.,
Hicks Leslie M.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00555
Subject(s) - chlamydomonas reinhardtii , microbiology and biotechnology , biochemistry , biology , chlamydomonas , oxidative phosphorylation , tor signaling , kinase , chemistry , gene , mutant
The target of rapamycin (TOR) kinase is a Ser/Thr kinase and master regulator , with roles in the regulation of nutritional sensing, protein translation, and autophagy, among others. In Chlamydomonas reinhardtii , a unicellular green algae and model organism for biofuel production and optimization, TOR has been linked to increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target is responsible for the elusive “lipid switch,” the protein or proteins responsible for increasing TAG formation under nutrient limitation and environmental stressors. However, when this lipid switch is activated, overall culture growth is arrested, truncating the alga’s ability to continuously produce TAGs at a level needed for widespread biofuel use. In order to engineer an effective strain for algal biofuels, the targets and signaling pathways of lipid regulatory networks must be determined. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems. Previous research has used TOR inhibitors to follow phosphorylation pathways and identify potential downstream targets of TOR but little work has uncovered the role of oxidative signaling in regulation. Since TOR inhibition results in an increase in reactive oxygen species analogous to that of nitrogen deprivation, the opportunity for redox control mechanisms is significant. This study inhibited TOR using AZD8055 and monitored reversible thiol oxidation while simultaneously tracking physiological changes. By pairing these data together, reversible thiol oxidation was related to nearly every major metabolic pathway in the cell, including TAG accumulation, the tricarboxylic acid cycle, and protein translation. Additionally, reversible thiol oxidation corresponded to changes in photosynthetic activity measured through chlorophyll a fluorescence, showing a novel role for TOR regulation of photosynthesis. The delineation of redox‐controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes. Support or Funding Information This research was supported by a National Science Foundation CAREER award (MCB‐1552522) awarded to L.M.H and a National Science Foundation award (Cooperative Agreement OIA‐1458,952) to D.R.J.K. A.L.S. was funded by the NASA West Virginia Space Grant Consortium (NNX15AK74A and NNX15AI01H).