Characterization of Saccharomyces cerevisiae suppressor mutants devoid of the membrane lipid phosphatidylcholine

Abstract

Phosphatidylcholine (PC) is the most abundant membrane lipid in most eukaryotes and considered essential. The yeast double deletion mutant cho2opi3 lacks the methyltransferases converting phosphatidylethanolamine (PE) to PC. As a consequence, the cho2opi3 mutant is a choline auxotroph that relies on supplementation with choline for the synthesis of PC by the CDP-choline route. We isolated cho2opi3 suppressor clones that suppress the auxotrophy for choline, and show robust growth in the absence of choline or choline substitute. Analysis of the lipidome of cho2opi3S clones revealed that PC is below the detection limit after culture without choline; instead PE has become the most abundant phospholipid. The neutral lipid triacylglycerol strongly accumulates in the PC-freecho2opi3 suppressors, and accordingly increased number and size of lipid droplets were observed by electron microscopy. Importantly, the lipidome of the PC-free cho2opi3 suppressor strains reveals an overall shift to shorter average acyl chain length, which is thought to play a key role in maintaining membrane physical properties. PC-freecho2opi3 suppressors function properly in processes like the regulation of UASINO gene expression and endocytosis, but lost the ability to grow on non-fermentable carbon source indicative for mitochondrial dysfunction. A phospholipid: diacylglycerol acyltransferase (PDAT)was found to be essential for the suppression of choline auxotrophy. Whole genome sequencing of a subset of suppressor mutants suggested 2N-1 aneuploidy as mechanism underlying the suppression of choline auxotrophy, which was proven by engineering a corresponding 2N-1 cho2opi3 mutant. The rewiring of lipid metabolism resulting from the changed stoichiometry of gene expression in the aneuploid suppressor strains is proposed to enable PC-free life.