loading . . . A Genome-wide Visual Screen Identifies Lysophosphatidylcholine as Counter Spatial Regulator of DAG and Sterols in Yeast Membrane lipids are heterogeneously distributed across the bilayers of cellular membranes. Cytosolic-facing pools of diacylglycerol (DAG) in the yeast Saccharomyces cerevisiae are enriched at both ends of the endomembrane system from the vacuolar membrane to the polarized plasma membrane (PM) of buds. However, how this distribution is maintained remains unknown. Using a genome-wide DAG biosensor screen in yeast, we identify regulators of DAG spatial distribution, enriched in proteins involved in vesicle or lipid transport and in phospholipid or sterol metabolism. A subset of mutants exhibited DAG mislocalization predominantly to the PM, with the most severe phenotype linked to a mutant of a predicted lipase we have named Drl1 (DAG redistribution lipase 1). Reversion of this phenotype required both enzymatic activity and the presence of an intrinsically disordered carboxy-terminal domain. Lipidomic analysis revealed that loss of Drl1 increased cellular lysophosphatidylcholine (LysoPC) levels. Remarkably, we find that supplementing cells with a non-metabolizable LysoPC analogue replicated the mutant DAG phenotype, implicating LysoPC as a novel spatial regulator of DAG. High-resolution imaging suggests that LysoPC reduces the PM sterol pool resulting in DAG expansion into new PM territories. More globally, our work expands the known interplay between various lipids and their co-regulation to maintain accurate membrane properties. ### Competing Interest Statement The authors have declared no competing interest. Natural Sciences and Engineering Research Council, https://ror.org/01h531d29, 2023-04320 Alberta Innovates, AI 242506270 Canadian Institutes of Health Research, https://ror.org/01gavpb45, MOP-142403 Universidad de la Ciudad de Buenos Aires, UBACYT 20020190100122BA Chan Zuckerberg Initiative (United States), 2023-331952 Weizmann Institute of Science, https://ror.org/0316ej306 https://www.biorxiv.org/content/10.64898/2026.01.27.702077v1
