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

Bruno L Bozaquel-Morais,Thiago M Venâncio,Monica Montero-Lomeli,Claudio A Masuda,Thiago Pacheco-Rosa,Juliana B Madeira,Michael Polymenis

doi:10.1371/journal.pone.0169682

Bruno L Bozaquel-Morais, Thiago M Venâncio + Show 5 more

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https://doi.org/10.1371/journal.pone.0169682

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Abstract

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.

Highlights

Cytosolic acetyl-CoA carboxylase (ACCase) plays a key role in lipid metabolism. This enzyme catalyses the carboxylation of acetyl-CoA, producing malonyl-CoA, a critical intermediate in fatty acid biosynthesis
As our main goal was to identify Acc1p interactors, we addressed if our results could be biased by the inhibition of a second acetyl-CoA carboxylase, Hfa1p, localized in the mitochondrial matrix
In sor1Δ sor2Δ and sor3Δstrains, no significant differences in triacylglycerol or steryl ester levels were observed. This result was unanticipated; we investigated the length of the acyl groups in the total cellular lipid fractions by gas chromatography/mass spectrometry (GC/MS) (Fig 6C, Table 2), as it has been recently shown that such parameter is affected by the activity of Acc1p [10,33]

Summary

Introduction

Cytosolic acetyl-CoA carboxylase (ACCase) plays a key role in lipid metabolism. This enzyme catalyses the carboxylation of acetyl-CoA, producing malonyl-CoA, a critical intermediate in fatty acid biosynthesis. In Saccharomyces cerevisiae, two ACCase isoforms were found, Acc1p, located in the cytoplasm, and Hfa1p, located in the mitochondrial matrix and less studied. Acc1p is essential in all organisms Even the supplementation of growth medium with longchain fatty acids does not reverse the lethality of its deletion in yeast [1]. PLOS ONE | DOI:10.1371/journal.pone.0169682 January 11, 2017

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