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

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

<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

Screening is not biased by Hfa1p.

<p>(A) Alignment of Hfa1p and Acc1p biotin carboxylase (BC) domain sequences. Soraphen A binds to the BC domain of Acc1p blocking its oligomerization [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref020" target="_blank">20</a>]. The residues (18) that were previously shown to interact with soraphen A in Acc1p BC domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref020" target="_blank">20</a>] are marked in yellow. The regulatory serine-77 is marked in red. Note that Hfa1p translation begins at a non-canonical (Ile) start codon in position -71 relative to the annotated initial methionine, marked in black [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref026" target="_blank">26</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169682#pone.0169682.ref027" target="_blank">27</a>]. (B) Soraphen A sensitivity in fermentative and respiratory substrates. WT strain was grown for 48 h in liquid YP medium containing 2% glucose (YPD) or 2% glycerol (YPGly) in the presence of increasing concentrations of soraphen A. Soraphen A IC₅₀ values were determined as described in Methods section. Results are the means of four independent experiments ± standard deviations (non-paired t-test; ns, non-significant).(C) The screen does not select negative interactors of <i>HFA1</i>. <i>hfa1Δ</i> strain shows no growth defects when grown under standard conditions in YPD; however, cell growth is impaired upon deletion of gene <i>A</i>, a negative interactor. If Hfa1p would be inhibited by soraphen A, a strain with a deletion of gene <i>A</i> (or any negative interactor of <i>HFA1</i>) would be more sensitive to the drug. That was not the case as only 3 out of 50 negative interactors were identified in the screen (<i>CPT1</i>, <i>PBS2</i> and <i>TPS2</i>), however they were filtered in subsequent analysis.</p

Characterization of neutral lipids, acyl lengths and soraphen A sensitivity of <i>sor1Δ</i>, <i>sor2Δ</i> and <i>sor3Δ</i> mutants.

<p>For lipid analysis, <i>SOR</i> null mutants and WT (BY4741) strains were grown in YPD. WT was also grown in the presence of a sub-lethal concentration of soraphen A (0.1 μg/ml). After 24 h, the total lipids were extracted and the lipid profile was determined by TLC (A,B) or GC/MS (C). Triacylglycerol (A) and sterol ester (B) contents were estimated from TLC plates by densitometry and were normalised to the values obtained for WT grown in YPD. (C) C18:0 and C16:0 extracted from total lipids were quantified and the C18:0/C16:0 ratio of each strain is shown. Results presented in (A), (B) and (C) are means of three independent experiments ± standard deviations (non-paired t-test; **, P < 0.01; *, P < 0.05; ns, non-significant). (D) Soraphen IC₅₀ values for single and <i>sorXΔsnf1Δ</i> mutants were determined as described in Methods section. As controls, <i>lys2Δ</i> and <i>snf1Δ</i> strains were employed. Results are the means of at least four independent experiments ± standard deviations (non-paired t-test; **, P < 0.01; *, P < 0.05; ns, non-significant). (E) Strains transformed with pSNF1 (pYC::SNF1-3HA) were grown in liquid YPD medium to log-phase and proteins were extracted for western blot analysis. Blots were probed with anti-phospho AMPK antibody (upper panel) or anti-hemagglutinin antibody (lower panel). HA-tagged Snf1p and endogenous Snf1p in phosphorylated form are designated as ^PSnf1-3HA and ^PSnf1, respectively. Total tagged Snf1p is designated as Snf1-3HA.</p

Candidate Acc1p interactors with their respective soraphen A sensitivity scores.

<p>Candidate Acc1p interactors with their respective soraphen A sensitivity scores.</p