Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets

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 d...

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Bibliographic Details
Published in:The New phytologist 2019-01, Vol.221 (1), p.247-260
Main Authors: Werth, Emily G., McConnell, Evan W., Couso Lianez, Inmaculada, Perrine, Zoee, Crespo, Jose L., Umen, James G., Hicks, Leslie M.
Format: Article
Language:English
Subjects:
Algae
AZD8055
Biosynthesis
Carotenoids
Carotenoids - metabolism
Cell growth
Chlamydomonas
Chlamydomonas reinhardtii
Chlamydomonas reinhardtii - drug effects
Chlamydomonas reinhardtii - genetics
Chlamydomonas reinhardtii - metabolism
Cluster Analysis
Homology
Identification
Inhibition
Kinases
Lycopene
Mechanistic Target of Rapamycin Complex 1 - antagonists & inhibitors
Mechanistic Target of Rapamycin Complex 1 - metabolism
Mineral nutrients
Morpholines
Mutation
Naphthyridines
Nutrients
Organic chemistry
Phosphoproteins - metabolism
phosphoproteomics
Phosphorylation
Phosphorylation - drug effects
Photosynthesis
Plant Proteins - metabolism
Protein Kinase Inhibitors - pharmacology
Proteins
Rapamycin
Signal transduction
Signaling
Sirolimus - pharmacology
Substrates
target of rapamycin (TOR)
TOR protein
TOR Serine-Threonine Kinases - antagonists & inhibitors
TOR Serine-Threonine Kinases - metabolism
Torin1
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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, AZD8055 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 RPS6 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 TORC1 inhibition and carotenoid production is under TOR control in algae.
ISSN:0028-646X
1469-8137
DOI:10.1111/nph.15339