Abstract
Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP7 and InsP8 ) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an invivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP7 and InsP8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses.
Highlights
Post-translational modifications (PTMs) rapidly regulate major cellular processes such as transcription, translation and metabolism
We previously demonstrated that phosphorylation of RPS6 on Ser245 is a readout of target of rapamycin (TOR) activity in Chlamydomonas (Couso et al, 2020)
Vip1-1 showed a significant decrease in phosphorylation level of RPS6 Ser245 (P-RPS6) compared with WT (Fig. 1a,b) after 30 min of Rap treatment that was more pronounced after 60 min (Fig. 1b,c) indicating a faster de-phosphorylation in vip1-1 compared with WT
Summary
Post-translational modifications (PTMs) rapidly regulate major cellular processes such as transcription, translation and metabolism Understanding these events is essential to map the complex signalling networks mediated by master regulators such as target of rapamycin (TOR) kinase (Soulard et al, 2010; Yu et al, 2011; Robitaille et al, 2013; Roustan & Weckwerth, 2018; Van Leene et al, 2019; Werth et al, 2019; Scarpin et al, 2020). The most common PP-InsPs arise from conversion of InsP6 to 1-diphosphoinositol 2,3,4,5,6-pentakisphosphate (1-InsP7 or 1PP-InsP5), 5diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7 or 5PPInsP5) and 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8 or 1,5(PP)2-InsP4) (Supporting Information Fig. S1), in
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