Balancing bilayer and non-bilayer lipids in yeast: mechanism and players
09 / 2010 - 09 / 2014
This research proposal focuses on the regulation of membrane lipid composition. Lowering the content of the major membrane lipid constituent phosphatidylcholine (PC) in the yeast Saccharomyces cerevisiae has revealed a novel regulatory mechanism for membrane lipid composition. We made this discovery in a yeast cho2opi3 mutant, in which synthesis of PC through methylation of phosphatidylethanolamine (PE) is ablated, and de novo synthesis of PC depends on the supply of choline from the medium. When logarithmically growing cho2opi3 (pem1pem2) cells were deprived of choline, cell growth continued at wild type rate for 4 generations. In this time window the level of PE strongly increased at the expense of PC, which was accompanied by a large shift in cellular acyl chain composition with the acyl chains becoming shorter and more saturated. The change in acyl chain profile was largest in PE, and was found to suppress the non-bilayer propensity of this lipid. The concomitant changes in acyl chain length and degree of saturation could not be reconciled with any known mechanism regulating membrane lipid composition in yeast. Hence, membrane intrinsic curvature, which depends on the relative proportions of bilayer and non-bilayer preferring phospholipids, was identified as novel physical membrane property determining membrane lipid composition in the model eukaryote baker?s yeast. Previously, regulation of membrane intrinsic curvature was demonstrated in prokaryotes, where it is essential for viability. The molecular details of this regulatory mechanism including the molecules that sense and transmit the changes in membrane lipid composition remain obscure. Here we aim to elucidate the mechanism and to identify the gene products that enable yeast to adapt its acyl chain composition in response to changes in PC and PE content. The robust, reversible changes in acyl chain composition that can be induced in cho2opi3 cells by deprivation and back-addition of choline, render this system uniquely suited to investigate the molecular mechanism(s) underlying the regulation of membrane intrinsic curvature. Suppressor genes that alleviate the growth defect of cho2opi3 cells under conditions of choline limitation will be identified in genome-wide genetic screens. The first screen will employ synthetic genetic array technology and the second will be based on suppression by overexpression. Subsequently, genes of interest emerging from the screens and genes already known to be involved in (the regulation of) fatty acid synthesis and desaturation, will be examined for their requirement in adapting the acyl chain composition. The required gene products will then be analyzed for mRNA and protein expression levels, posttranslational modification and enzyme activity during depletion and restoration of PC contents in cho2opi3 cells, to reveal where regulation occurs. The results will be integrated into hypotheses accounting for the mechanism regulating the balance between bilayer and non-bilayer lipids that will be tested experimentally.