A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis

Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone

A Chemogenomic Screen Reveals Novel Snf1p/AMPK Independent Regulators of Acetyl-CoA Carboxylase.

Acetyl-CoA carboxylase (Acc1p) is a key enzyme in fatty acid biosynthesis and is essential for cell viability. To discover new regulators of its activity, we screened a Saccharomyces cerevisiae deletion library for increased sensitivity to soraphen A, a potent Acc1p inhibitor. The hits identified in the screen (118 hits) were filtered using a chemical-phenotype map to exclude those associated with pleiotropic drug resistance. This enabled the identification of 82 ORFs that are genetic interactors of Acc1p. The main functional clusters represented by these hits were "transcriptional regulation", "protein post-translational modifications" and "lipid metabolism". Further investigation of the "transcriptional regulation" cluster revealed that soraphen A sensitivity is poorly correlated with ACC1 transcript levels. We also studied the three top unknown ORFs that affected soraphen A sensitivity: SOR1 (YDL129W), SOR2 (YIL092W) and SOR3 (YJR039W). Since the C18/C16 ratio of lipid acyl lengths reflects Acc1p activity levels, we evaluated this ratio in the three mutants. Deletion of SOR2 and SOR3 led to reduced acyl lengths, suggesting that Acc1p is indeed down-regulated in these strains. Also, these mutants showed no differences in Snf1p/AMPK activation status and deletion of SNF1 in these backgrounds did not revert soraphen A sensitivity completely. Furthermore, plasmid maintenance was reduced in sor2Δ strain and this trait was shared with 18 other soraphen A sensitive hits. In summary, our screen uncovered novel Acc1p Snf1p/AMPK-independent regulators

Expression of the glucose transporter HXT1 involves the Ser-Thr protein phosphatase Sit4 in Saccharomyces cerevisiae

We studied the effect of the loss of the Ser-Thr protein phosphatase Sit4, an important post-translational regulator, on the steady-state levels of the low-affinity glucose transporter Hxt1p and observed a delay in its appearance after high glucose induction, slow growth, and diminished glucose consumption. By analyzing the known essential pathway necessary to induce Hxt1p, we observed a partial inhibition of casein kinase I activity. In both WT and sit4Δ strains, the transcript was induced with no significant difference at 15 min of glucose induction; however, after 45 min, a clear difference in the level of expression was observed being 45% higher in WT than in sit4Δ strain. As at early time of induction, the HXT1 transcript was present but not the protein in the sit4Δ strain we analyzed association of HXT1 with ribosomes, which revealed a significant difference in the association profile; in the mutant strain, the HXT1 transcript associated with a larger set of ribosomal fractions than it did in the WT strain, suggesting also a partial defect in protein synthesis. Overexpression of the translation initiation factor TIF2/eIF4A led to an increase in Hxt1p abundance in the WT strain only. It was concluded that Sit4p ensures that HXT1 transcript is efficiently transcribed and translated thus increasing protein levels of Hxt1p when high glucose levels are present. © 2012 Federation of European Microbiological Societies

<i>SOR2</i> deletion reduced Acc1p activity and plasmid loss are not directly linked.

<p>(A) Strains transformed with pYC (empty vector) or pSNF1 (pYC::SNF1-3HA), both with <i>URA3</i> as a genetic marker, were grown for 48 h in liquid SD medium in the presence of selective pressure (without uracil supplementation). The percentage of plasmid-carrying cells was quantified by plating 200 cells of each culture on SC agar +ura or–ura, and colony counting after 3 da. For WT + pYC and WT + pYC:SNF1, n = 1; for <i>sor2Δ</i> + pYC and <i>sor2Δ</i> + pYC:SNF1, n = 3 (three different isolated clones were tested). (B) WT strain carrying pYC (empty vector) was grown for 48 h in liquid SD medium in the presence of selective pressure (without uracil), with the indicated concentrations of soraphen A. Plasmid stability was assessed as in (B) and the data are presented as the means from two experiments.</p

Acyl composition in total lipid extracts of WT and <i>SOR</i> null mutants after 24 h growth in rich YPD medium, as determined by GCMS.

<p>Acyl composition in total lipid extracts of WT and <i>SOR</i> null mutants after 24 h growth in rich YPD medium, as determined by GCMS.</p

<i>ACC1</i> transcriptional levels do not correlate with soraphen A sensitivity.

<p>Mutant strains with deletion of genes encoding transcriptional factors (TFs) that affect <i>ACC1</i> transcript levels were retested for soraphen A sensitivity as before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.g003" target="_blank">Fig 3</a>). Published <i>ACC1</i> transcript levels in these mutants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref032" target="_blank">32</a>] are shown. TFs mutant strains are marked in green or red, depending on whether <i>ACC1</i> transcription was down-regulated or up-regulated, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref031" target="_blank">31</a>]. TF mutant strains identified by our screen but with unaltered <i>ACC1</i> transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref030" target="_blank">30</a>] are marked in black. The <i>lys2Δ</i> strain was employed as a control. The results are representative of four independent experiments. ^{1, 2} Two <i>yjl206cΔ</i> strains were present in our collection and both were tested.</p

Chemical phenotype (CP) analysis identifies hits that according to rank plot analysis are specifically associated with soraphen A treatment.

<p>(A) CP network is a bipartite hybrid network comprising ORFs and drugs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref015" target="_blank">15</a>]. An ORF is connected to a drug when its deletion increases sensitivity to that drug. LipCP network was extracted by choosing drugs that target the lipid metabolism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.s004" target="_blank">S3 Table</a>) and their connected ORFs from the CP network (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.s005" target="_blank">S4 Table</a>). (B) ORFs identified among our final hits (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.s003" target="_blank">S2 Table</a>), plotted according to their relative connectivity in both CP and LipCP networks. The score represents the number of drug connections of an ORF in each network, normalised so that the most connected ORF scores 1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.s006" target="_blank">S5 Table</a>). Quadrants are numbered as shown. ORFs that are located in the third quadrant and with degree lower than 0.4 in both networks, represented by dotted lines, were considered to be primarily associated with soraphen A treatment. Final hits are marked as black dots. Blue circles represent ORFs that were missed by the screen but were added in later after individual re-testing (<i>SPT4</i>, <i>SFP1</i> and <i>TUP1</i>; please refer to the text).</p