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Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets
Author(s) -
Werth Emily G.,
McConnell Evan W.,
Couso Lianez Inmaculada,
Perrine Zoee,
Crespo Jose L.,
Umen James G.,
Hicks Leslie M.
Publication year - 2019
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.15339
Subject(s) - chlamydomonas reinhardtii , chlamydomonas , phosphoproteomics , biology , phosphorylation , kinase , microbiology and biotechnology , biochemistry , protein phosphorylation , protein kinase a , mutant , gene
Summary 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, AZD 8055 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 RPS 6 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 TORC 1 inhibition and carotenoid production is under TOR control in algae.