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 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

Plasmids Used in This Study.

<p>Plasmids Used in This Study.</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

TORC1 antagonizes Nem1 phosphorylation.

<p>(A, B) SDS-PAGE (A) and Phos-tag phosphate-affinity gel electrophoresis (B) analyses of plasmid-encoded Nem1-HA₃ in exponentially growing <i>nem1</i>Δ cells treated with rapamycin (RAP) for the indicated times. The levels of Pgk1 served as loading controls in (A). (C) Phosphorylation pattern analysis of Nem1-PtA on Phos-tag gels. Plasmid-encoded Nem1-PtA was purified from exponentially growing (EXP) and rapamycin-treated (RAP; 30 min) <i>nem1</i>Δ cells and treated with (+), or without (−), alkaline phosphatase (AP) in the absence (−), or presence (+), of phosphatase inhibitors (PPI). P0, P1, and P2 (in B and C) denote 3 differentially phosphorylated Nem1-HA₃ or Nem1-PtA isoforms.</p

Strains Used in This Study.

<p>Strains Used in This Study.</p