Abstract
To identify proteins with a functional role in lipid metabolism and homeostasis we designed a high-throughput platform for high-content lipidomic screening of yeast mutant libraries. To this end, we combined culturing and lipid extraction in 96-well format, automated direct infusion nanoelectrospray ionization, high-resolution Orbitrap mass spectrometry, and a dedicated data processing framework to support lipid phenotyping across hundreds of Saccharomyces cerevisiae mutants. Our novel approach revealed that the absence of genes with unknown function YBR141C and YJR015W, and the transcription factor KAR4 precipitated distinct lipid metabolic phenotypes. These results demonstrate that the high-throughput shotgun lipidomics platform is a valid and complementary proxy for high-content screening of yeast mutant libraries.
Highlights
Genetic and biochemical studies using the yeast Saccharomyces cerevisiae have been instrumental in elucidating the blueprint of lipid metabolism and defining the physiological functions of lipids.[6,7] Early efforts have pinned lipid metabolism to the framework of global metabolism as illustrated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway resource.[8]
These results show that combining functional genomic workflows and high-content lipidomic profiling can be a powerful proxy for identifying regulators of global lipid metabolism
We compiled a deletion library in 96-well format covering 178 strains divided into three groups: 168 deletion mutants of genes encoding transcriptional regulators; 8 deletion mutants of genes encoding proteins with unknown function and predicted to be involved in lipid homeostasis; and control strains including the BY4742 reference and the elo2D mutant with defective fatty acid elongase activity[36] (Table S1, Electronic supplementary information (ESI)†)
Summary
Genetic and biochemical studies using the yeast Saccharomyces cerevisiae have been instrumental in elucidating the blueprint of lipid metabolism and defining the physiological functions of lipids.[6,7] Early efforts have pinned lipid metabolism to the framework of global metabolism as illustrated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway resource.[8]. The circuitry in S. cerevisiae includes the transcriptional regulators Opi1p, Ino2p, Ino4p and Zap1p that control the expression level of a subset of proteins required for glycerophospholipid metabolism.[9] In addition, the circuitry includes the regulators Mga2p and Spt23p, which are involved in controlling the expression of the fatty acid desaturase OLE1.10 Notably, regulatory mechanisms controlling most of enzymes in the lipid metabolic network are still poorly understood. This raises the question of how the expression levels of enzymes involved in, for example, sphingolipid and sterol lipid metabolism are controlled
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