Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

Abstract

The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP 6 ) to produce the low abundance signaling molecules InsP 7 and InsP 8 . Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP 7 and InsP 8 , both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation.

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

“…Moreover, although commercial anti‐phospho S6K antibodies have been shown to detect phospho‐S6K in plants (Xiong & Sheen, ; Ahn et al ., ), they have detected a signal in Chlamydomonas neither in the present authors’ hands (Fig. S6) nor in another study (Couso et al ., ), thus limiting our ability to independently validate Chlamydomonas TOR substrate phosphopeptides. However, Chlamydomonas ribosomal protein S6 (RPS6, Cre09.g400650.t1.2), a predicted target of S6K, showed a 2.1‐fold decrease in phosphorylation on Thr127 following Torin1 treatment (Fig.…”

Eukaryotic Phosphate Homeostasis: The Inositol Pyrophosphate Perspective

“…With this in mind, the altered mitochondria functionality of ip6k1 -/-mice might explain the inability of these mice to become obese [64]. Further evidence of the evolutionarily conserved ability of PP-IPs to regulate primary metabolism comes from the recent metabolomics analysis of the unicellular green alga Chlamydomonas reinhardtii, which demonstrated the importance of PP-IPs in regulating tricarboxylic acid (TCA) cycle and fatty acid synthesis [65] Interestingly, kcs1 yeast, while possessing an adenylate energy charge close to 1, also have an absolute increase in the three adenine nucleotides (AMP, ADP and ATP) [61]. The nucleotide pools might therefore play an important, but poorly characterised, role in buffering cellular phosphate.…”

Reciprocal regulation of photosynthesis and mitochondrial respiration by TOR kinase in Chlamydomonas reinhardtii

“…As expected, TOR kinase plays a central role in growth, metabolism, ER stress, autophagy, carbon/nitrogen balance, and lipid accumulation in C. reinhardtii (Juppner et al, ; Mubeen et al, ; Perez‐Perez, Couso, & Crespo, ). The role of TOR kinase in cellular phosphate metabolism was also uncovered in TOR hypersensitive mutants revealing lower levels of InsP 6 and InsP 7 (inositol phosphates) (Couso et al, ). Counterintuitively, one of the recent studies concludes that TOR kinase inhibition positively regulates nitrogen assimilation leading to increased ammonium uptake and amino acid biosynthesis (Mubeen et al, ).…”