Who let the DAG's out? A genome wide survey in yeast identifies a novel putative lipase related to diacylglycerol dynamics

Abstract

Diacylglycerol (DAG) was the first lipid second messenger to be discovered in the canonical activation pathway of protein kinase C (PKC). The informational outcomes of DAG seem to be intimately connected with its spatial distribution within the cell necessitating methods to track its different cellular pools. We have previously developed a fluorescent DAG probe based on the C1 domain of PKCδ to monitor cytoplasmic facing pools of DAG in budding yeast using fluorescence live microscopy. Two pools of DAG were identified in both the vacuole and at sites of polarized growth. To better understand how DAG distribution is regulated, a genome-wide, high-throughput imaging screen surveying single knockout and hypomorphic yeast collections expressing the DAG probe was conducted. From the ⁓6,000 strains imaged, we have identified a discrete group of mutant strains where DAG localized predominantly to the plasma membrane. The most extreme phenotype in this category was observed in a mutant producing a truncated version of an uncharacterized putative lipase. It was determined that the abnormal DAG phenotype depended on both the activity of the enzyme and its carboxy-end, as these were both requisites for reversion of the phenotype. Lipidomic analysis revealed that ablation of this lipase increases cellular levels of Lysophosphatidylcholine (LysoPC). Addition of a non-metabolizable LysoPC analogue reproduced the mutant DAG phenotype suggesting a causal relationship between LysoPC levels and DAG. This study aims to tackle one of the ~700 functionally uncharacterized genes in S. cerevisiae while also setting the groundwork for understanding DAG dynamics and regulation.