A chemical genomic screen identifies novel genetic interactors of the methylation-dependent de novo pathway of phosphatidylcholine biosynthesis in Saccharomyces cerevisiae

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in most eukaryotic cell and organelle membranes, and cells must regulate its synthesis and interorganelle transport to maintain correct membrane compositions and biophysical properties. Our knowledge of genes encoding enzymes of PtdCho biosynthesis is largely complete, and a great deal is understood about their localization and regulation, however our understanding of molecular mechanisms regulating PtdCho biosynthesis and trafficking remains incomplete. To identify genes that show epistatic relationships with the methylation pathway of PtdCho biosynthesis, we performed a chemical-genomic screen of a Saccharomyces cerevisiae gene-deletion mutant collection using a phosphatidylethanolamine (PtdEtn) methyltransferase inhibitor, 2-hydroxyethylhydrazine (HEH). HEH functions by selectively inhibiting the PtdEtn methyltransferase enzymes Pem1p/Cho2p and Pem2p/Opi3p. We demonstrate that the addition of exogenous choline or lyso-phosphatidylcholine can recover HEH-mediated growth inhibition, and used this finding to design a functional-genomic screen to identify genes which, when deleted, render the strain unable to grow when the methylation pathway is partially inhibited. We now report the identification of 410 S. cerevisiae gene deletion mutants that exhibit HEH hypersensitivity, and identify among those a core set of 21 genes that are known to epistatically interact with genes encoding enzymes of the PtdEtn methylation pathway. This gene set was enriched in functions relating to glycerolipid and sterol biosynthesis and their regulation, the high-osmolarity glycerol (HOG) pathway, and genes involved in chromatin remodeling and transcriptional regulation. These results demonstrate that PtdCho produced by any one of the Kennedy pathway, methylation pathway, or acyltransferase pathway can maintain necessary cellular PtdCho compositions, but that disruption of any one of these pathways leads to epistatic interactions with non-overlapping subsets of genes, thus providing new insights on the specific functions of these pathways. The design and implementation of this screening strategy establishes HEH as a useful tool for specific inhibition of the methylation pathway in high-throughput functional-gnomic screens, which will facilitate further studies on the synthesis, transport, and function of PtdCho.