Mechanisms regulating the acyl chain composition of membrane lipids in S. cerevisiae

Abstract

The goal of the research described in this thesis was to identify the genes/enzymes involved in acyl chain remodeling of the major membrane lipid phosphatidylcholine (PC) in the model eukaryote S. cerevisiae. A database search yielded 75 candidates with (potential) phospholipase, acyltransferase or transacylase activity. The corresponding deletion strains were screened for the molecular species profiles of PC and phosphatidylethanolamine (PE), yielding 10 candidate strains. Using stable isotope labeling followed by analysis by mass spectrometry, the evolution of the species profile of PC newly synthesized via PE methylation was monitored in pulse-chase experiments, revealing that 6 strains displayed aberrant PC remodeling. Deletion of the SCT1/GAT2 gene coding for a glycerol-3-phosphate acyltransferase, led to the most pronounced change in PC remodeling. In-depth study into the role of Sct1p in regulating lipid acyl chain composition showed that Sct1p regulates fatty acyl chain desaturation, as deletion and overexpression of SCT1 dramatically change the acyl chain composition. We showed that it does so by competing with the fatty acid desaturase Ole1p for the common substrate C16:0-CoA in co-overexpression experiments of OLE1 and SCT1. A new paradigm for the regulation of fatty acid desaturation is presented in which an acyltransferase sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by a fatty acid desaturase. Sct1p activity is regulated by phosphorylation. The level of Sct1p phosphorylation increases in response to the presence of C16:0 acyl chains in the culture medium, demonstrating the physiological relevance of these findings. Overexpression of Sct1p dramatically increases the cellular C16:0 content, and this excess C16:0 is incorporated into all glycerophospholipids: phosphatidic acid (PA), phosphatidylserine (PS), PE, PC, phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL). The extra C16:0 is incorporated into PC via both biosynthesis routes, and via enhanced remodeling. The increase in the extent of remodeling allowed a much more sensitive detection of PC acyl chain remodeling. Taking advantage of Sct1p overexpression we showed that remodeling occurs at both the sn-1 and sn-2 position of the glycerol backbone, and that the phospholipase B Plb1p is required for efficient remodeling. Plb2p and Lro1p were not required for PC acyl chain remodeling. Plb1p is the first enzyme that is shown to be directly involved in PC acyl chain remodeling, most probably by hydrolyzing both acyl chains. The role of PC as potential acyl donor of Taz1p, a CL remodeling transacylase, was also investigated. Analysis of the molecular species profiles of steady state and newly synthesized glycerophospholipids indicates that PS and/or PE rather than PC is the preferred acyl donor of Taz1p.