TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved

Etude de la voie de synthèse du monogalactosyldiacylglycérol dans les tissus photosynthétiques (régulation de MGD1 par l'acide phosphatidique)

Le développement des chloroplastes dépend de la synthèse du galactoglycérolipide monogalactosyldiacylglycérol (MGDG). Le MGDG est formé dans les chloroplastes par l enzyme MGD1 par transfert du galactose de l UDP-galactose sur le diacylglycérol (DAG), dont une partie provient de l'hydrolyse des phospholipides extraplastidiaux par des phospholipases D (PLD) via l'acide phosphatidique (AP). Dans la voie de synthèse, cette étape semble importante puisque l'AP est le précurseur général des glycérolipides et un messager secondaire clé. Dans cette thèse, nous analysons le rôle de l'AP dans la synthèse du MGDG. Nous montrons que : 1) la teneur en AP de cultures cellulaires d Arabidopsis affecte la quantité de MGDG formé par son action stimulatrice sur l'activité MGD, 2) l'AP active également le niveau de synthèse de MGDG dans des homogénats de feuilles et dans de l'enveloppe de chloroplastes isolée, et 3) l AP active la protéine recombinante MGD1 de manière allostérique. Nous montrons que PLD?2 est localisée sur le tonoplaste et est potentiellement impliquée dans la régulation du niveau d activité MGDG synthase. Par ailleurs, nous mettons en évidence que le phosphatidylglycérol (PG) est un deuxième activateur allostérique de MGD1. La comparaison du comportement de MGD1 en présence d AP ou de PG indique que l AP et le PG activent MGD1 par des mécanismes différents et se fixent sur MGD1 en des sites distincts. L analyse de mutants ponctuels de MGD1 permet d identifier des résidus impliqués dans l activation de MGD1 par le PG. Suite à ce travail, nous proposons un modèle de régulation de la synthèse du MGDG dans les cellules foliaires.Chloroplast development is dependent on the synthesis of monogalactosyldiacylglycerol (MG DG) galactoglycerolipid. MGDG synthesis occurs in chloroplasts and is catalyzed by MGD1 enzyme which transfers galactose from UDP-galactose to diacylglycerol (DAG) . DAG is for one part issued from phospholipid hydrolysis by phospholipases 0 (PLO) via phosphatidic acid (PA). This step seems important for this synthetic pathway because PA is a general precursor of ail glycerolipides but also a key signalling molecule. ln this thesis we analyze the role of PA in MGDG synthesis. We show that : 1) PA level in Arabidopsis suspension cells affects MGDG level by stimulating MGD activity, 2) PA also stimulates MGDG synthesis in leaf homogenates and in isolated chloroplast envelope, and 3) PA allosterically activates MGD1 recombinant protein. We show that PLD<2 is localized on the tonoplast membrane and is potentially involved in the regulation of MGDG synthase activity. Furthermore, we show that PG is a second allosteric activator of MGD1. Comparison of PA and PG effects on MGD1 demonstrates that PA and PG activates MGD1 through different mechanisms and have distinct binding sites on MGD1. Analysis of MGD1 proteins exhibiting point mutations allows identification of residues involved in MGD1 activation by PG. Based on this work, we propose a regulatory model for MGDG synthesis in leaf.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Glycerolipid biosynthesis and chloroplast biogenesis

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Lipid trafficking in plant photosynthetic cells

International audienceEach of the various membranes in plant cells has a specific glycerolipid composition, which is kept relatively stable in different cells and different plants. Lipid homeostasis effectors, particularly lipid transporters, remain largely uncharacterized. Recent progresses in the field rely on the analysis of chloroplast lipid homeostasis as a model of choice. Galactolipids are the main lipids of chloroplast membranes. Galactolipid synthesis occurs in the chloroplast envelope membranes and depends on the fine exchange of lipid intermediates between the envelope membranes. This synthesis is also highly dependent on supply of lipid precursors synthesized in the endoplasmic reticulum membranes. Phosphatidic acid is an important lipid intermediate that is generated in the envelope but also in various extraplastidic membranes belonging to the endosomal network. It was recently shown that extraplastidic phosphatidic acid is one of the galactolipid precursors. As it is also a signalling molecule in plant cells, it could be a regulator of the lipid synthesis pathway. Trafficking of phosphatidic acid in the envelope is, therefore, a key step for synthesis of chloroplast lipids. After their completion, galactolipids are transferred to the thylakoids possibly through vesicles. The stability of the membrane lipid composition indicates a tight regulation at the subcellular level. This control is, however, modified when the plant is deprived of phosphate resulting in enrichment of digalactosyldiacylglycerol in mitochondrial membranes, the tonoplast and the plasma membrane. The transfers of specific lipids into and out of the chloroplast envelope, particularly the transfer of digalactosyldiacylglycerol to the mitochondria, are enhanced. The molecular mechanisms potentially involved in transport of these glycerolipids are surveyed

Role of phosphatidic acid in plant galactolipid synthesis

Phosphatidic acid (PA) is a precursor metabolite for phosphoglycerolipids and also for galactoglycerolipids, which are essential lipids for formation of plant membranes. PA has in addition a main regulatory role in a number of developmental processes notably in the response of the plant to environmental stresses. We review here the different pools of PA dispatched at different locations in the plant cell and how these pools are modified in different growth conditions, particularly during plastid membrane biogenesis and when the plant is exposed to phosphate deprivation. We analyze how these modifications can affect galactolipid synthesis by tuning the activity of MGD1 enzyme allowing a coupling of phospho- and galactolipid metabolisms. Some mechanisms are considered to explain how physicochemical properties of PA allow this lipid to act as a central internal sensor in plant physiology

ALA10, a phospholipid flippase, controls FAD2/FAD3 desaturation of phosphatidylcholine in the ER, and affects chloroplast lipid composition in Arabidopsis thaliana.

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TORC1 inhibits Pah1 function in part by preventing phosphorylation of Ser¹⁹⁵ in Nem1.

<p>(A) Phos-tag phosphate-affinity gel electrophoresis analysis of full length and schematically indicated truncated, plasmid-encoded Nem1-HA₃ variants in exponentially growing (RAP; 0 min) and rapamycin-treated (RAP; 30 min) wild-type cells. The two dark grey boxes in the N-terminal region denote membrane-spanning regions and the black stripe within the highly conserved C-terminal domain (grey box) indicates the position of the Nem1 catalytic site. (B) Phos-tag phosphate-affinity gel electrophoresis analysis of plasmid-encoded Nem1-HA₃ and Nem1^S195A-HA₃ in exponentially growing (RAP; 0 min) and rapamycin-treated (RAP; 30 min) <i>nem1</i>Δ cells. P0, P1, and P2 denote 3 differentially phosphorylated full-length (in [A] and [B]) or truncated (in [A]) Nem1-HA₃ isoforms. (C) Incorporation of radioactively labeled palmitic acid into triacylglycerol (TAG) was monitored in exponentially growing (EXP) and rapamycin-treated (RAP; 90 min) <i>nem1</i>Δ <i>PAH1-HA₃</i> cells that carried either an empty plasmid or a plasmid allowing the expression of PtA-tagged Nem1 or Nem1^S195A. Relevant genotypes of strains are indicated. (D) SDS-PAGE analysis of endogenously tagged Pah1-HA₃ from <i>nem1</i>Δ cells coexpressing, or not, plasmid-encoded PtA-tagged Nem1 or Nem1^S195A. Cells were either grown exponentially (RAP; 0 min) or treated with rapamycin (RAP) for the times indicated. Pah1-HA₃ levels were quantified, normalized with respect to the Adh1 loading control, and expressed in percent relative to the value at time point 0 (see numbers below the panels). Numbers represent means ± SD of three experiments. Relevant genotypes of strains are indicated. (E) Model for the role of TORC1 in controlling TAG synthesis in yeast. TORC1 indirectly regulates (dashed bar) the phosphorylation status of Ser¹⁹⁵ (and potentially other residues; indicated by the dashed arrow and the question mark) in Nem1 by activating or inhibiting hitherto unknown protein phosphatase(s) or kinase(s), respectively. Arrows and bars denote positive and negative interactions, respectively. For details, see text.</p

TORC1 has little impact on the interaction between Pah1 and the Nem1-Spo7 module.

<p>(A) Biochemical interaction between Nem1 and Pah1. Plasmid-encoded Dga1-PtA or Nem1-PtA was immunoprecipitated from extracts of Pah1-HA₃-expressing wild-type (lane 1) or <i>nem1</i>Δ cells (lanes 2–4), respectively, that were either grown exponentially (0 min) or treated with rapamycin (RAP) for the indicated times. Lysates (Input) and immunoprecipitates (PtA-Pulldown) were subjected to SDS-PAGE and immunoblots were probed with anti-HA or anti-IgG antibodies. WT and Δ denote wild-type and deleted version of <i>NEM1</i>, respectively. Numbers below the PtA-Pulldown blots indicate the relative amount of Pah1-HA₃ that bound to and was pulled down with Nem1-PtA (normalized to the samples of exponentially growing cells). (B) Biochemical interaction between Spo7 and Nem1/Pah1. Plasmid-encoded Spo7-PtA was immunoprecipitated from extracts of untreated (0 min) and rapamycin-treated (RAP; 30 min) <i>nem1</i>Δ <i>spo7</i>Δ <i>PAH1-HA₃</i> cells that coexpressed plasmid-encoded Nem1-HA₃. For details see (A). (C) The interaction between Nem1 and Pah1 requires Spo7. Plasmid-encoded Nem1-PtA was immunoprecipitated from extracts of exponentially growing, Pah1-HA₃-expressing <i>nem1</i>Δ (lane 2) or <i>nem1</i>Δ <i>spo7</i>Δ (lane 3) cells. Pah1-HA₃-expressing wild-type cells were used as control (lane 1). Please note that loss of Spo7 consistently resulted in decreased levels of Nem1. For details see (A). WT and Δ denote wild-type and deleted version(s), respectively, of the indicated gene(s). (D) The interaction between Spo7 and Pah1 does not require Nem1. Plasmid-encoded Dga1-PtA or Spo7-PtA was immunoprecipitated from extracts of exponentially growing, Pah1-HA₃-expressing <i>nem1</i>Δ (lane 1) or <i>nem1</i>Δ<i> spo7</i>Δ (lanes 2 and 3) cells, which coexpressed, or not, plasmid-encoded Nem1-HA₃. Please note that our anti-HA antibodies weakly cross-react with proteins that are present in cell lysates (indicated by the asterisk), but absent in the PtA-pulldown fractions. For details see (A). WT and Δ denote wild-type and deleted version(s), respectively, of the indicated gene(s). (E) Spo7 specifically interacts with both Nem1 and Pah1, while Nem1 only interacts with Spo7, but not with Pah1, when assayed in a split-ubiquitin membrane-based yeast two-hybrid assay. Interactions were tested by monitoring either growth on plates lacking adenine (-Ade), or β-galactosidase activities (in Miller units; numbers on the right of the panels represent the means of three independent experiments performed with exponentially growing cells) of cells expressing the indicated combinations of NubG-Spo7 or NubG-Nem1 and Nem1-Cub, Pah1-Cub, Spo7-Cub, or Mon1-Cub (control).</p

TORC1 inhibition activates Pah1 phosphatidate phosphatase via the Nem1-Spo7 protein phosphatase module.

<p>(A) Incorporation of radioactively labeled palmitic acid into triacylglycerol (TAG) was monitored in exponentially growing (EXP) and rapamycin-treated (RAP; 90 min) cells. Relevant genotypes of strains are indicated (WT, wild type). (B) Representative TLC plate showing radioactively-labeled, separated lipid samples from the experiment in (A) that were extracted from exponentially growing (RAP; −) and rapamycin-treated (RAP; +) WT, <i>pah1</i>Δ, and <i>nem1</i>Δ strains. STE, steryl esters; FFA, free fatty acids; DAG, diacylglycerol; MAG, monoacylglycerol; PL, phospholipids. (C) The combined levels of DAG and TAG were determined in rapamycin-treated (4 h) cells using a commercially available enzymatic kit and expressed in each case relative to the respective levels in exponentially growing cells. (D) Relative PAP activity in exponentially growing (EXP) and rapamycin-treated (RAP; 30 min and 60 min) cells. Results are presented as relative activities compared to the activity in exponentially growing <i>app1</i>Δ <i>dpp1</i>Δ<i> lpp1</i>Δ cells (defined as 1.0), which express Pah1 as only source of PAP activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104194#pone.0104194-Chae1" target="_blank">[44]</a>. Assays carried out in the presence of EDTA are indicated (+ EDTA). (E) Phos-tag phosphate-affinity gel electrophoresis and SDS-PAGE analyses of endogenously tagged Pah1-HA₃ in exponentially growing WT and <i>nem1</i>Δ cells treated with rapamycin (RAP) for the indicated times. The levels of Pgk1 served as loading controls. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104194#pone-0104194-g001" target="_blank">Figures 1A, 1C, and 1D</a>, each bar represents the mean ± SD of three experiments.</p