Novel Aspects of Phosphatidylcholine Metabolism

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Abstract Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cells. It serves as a structural component of cellular membranes and a reservoir of lipid second messengers. PC is made de novo by two primary routes: the CDP-choline branch of the Kennedy pathway and the PE methylation pathway. We recently characterized a third route in which the PC molecule is remodeled, as its fatty acyl groups are subject to a deacylation/reacylation pathway (PC-DRP). Importantly, alterations in PC synthesis, turnover and remodeling are associated with cellular malfunctions and disease states. PC-DRP involves complete deacylation of PC via phospholipases (Plb1, Nte1) that produce glycerophosphocholine (GPC) and successive acyl-CoA-dependent acyltransferase reactions that convert GPC à lysoPC à PC, as catalyzed by Gpc1 and Ale1. Gpc1, which catalyzes the committed step in the reacylation sequence and was discovered in our laboratory, provides the cell with i) a 2-step PC resynthesis route and ii) a means by which both PC acyl chains can be post-synthetically remodeled. Acyl chain content is crucial to cell function, as it affects fundamental membrane properties such as fluidity and curvature. In the previous grant period, we confirmed the role of Gpc1 in PC metabolism in S. cerevisiae and found that loss of Gpc1 results in a decrease in mono-unsaturated PC species and an increase in di-unsaturated PC species, thus confirming the role for PC-DRP in PC remodeling. The Unfolded Protein Response (UPR) is a signaling pathway responsive not only to unfolded proteins, but also ER bilayer stress. We have shown that Gpc1 is a key player in ER membrane bilayer homeostasis: its transcription is increased upon induction of the UPR and deletion of Gpc1 results in UPR induction. Strains bearing a deletion in GPC1 (gpc1D mutants) display other phenotypes, including decreased stationary phase viability and decreased sensitivity to growth at elevated temperature. Some of the observed phenotypes may be unrelated to acyltransferase activity, as Gpc1 also exhibits a lesser transacylase activity and, like other enzymes, may have cellular functions unrelated to catalysis. Gpc1 bears no sequence similarity to known acyltransferases or transacylases, and has been designated a new protein family (UniProtKB - P48236). Gpc1 homologs are lacking in vertebrates but are found in other organisms, including medically important pathogenic fungi. Our published finding that transport of the Gpc1 substrate, GPC, into C. albicans is required for full virulence of the organism, underlines the importance of examining enzymes, like Gpc1, that utilize GPC. Our objective is to use targeted mutagenesis to identify key amino acid residues required for Gpc1 acyltransferase activity. The catalytically-compromised mutant(s) will be used to elucidate the importance of acyltransferase activity to phenotypes associated with loss of the protein. In addition, we will interrogate the importance of the PC remodeling aspect of Gpc1 by examining its regulation in response to changes in membrane saturation. The research will be performed with the genetically tractable S. cerevisiae.