Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

Abstract

Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).

The glycerophosphocholine acyltransferase Gpc1 is part of a phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast

“…The glycerophosphodiesterase encoded by GDE1 can hydrolyze GPC to produce glycerol-3-phosphate and choline (14,19). Additionally, GPC can be acylated (likely at the sn-1 position) to LPC by Gpc1, a recently described acyltransferase (17). LPC can be converted to PC by Ale1, consistent with its role as a lysophospholipid acyltransferase (18,26).…”

“…We reported previously that Gpc1 had limited ability to utilize acyl-CoAs to acylate glycerophosphoethanolamine in an in vitro system (17). To probe the possibility that Gpc1 acylates glycerophosphoethanolamine to affect PE in vivo, we analyzed the PE molecular species.…”

“…Importantly, de Kroon and co-workers (22) have used stable-isotope pulse-chase analysis coupled with MS to prove the postsynthetic formation of more saturated PC species as a function of time and demonstrated the involvement of Plb1. Prior to the identification of Gpc1 activity (16,17), PC remodeling was believed to involve the removal of a single acyl chain at the sn-2 position, followed by reacylation by a LPC acyltransferase, a cycle first described by Lands (44). Once LPC is formed, it can be acylated to PC by Ale1 (18,26,45), with some publications indicating that Ale1 prefers incorporation of an unsaturated acyl-CoA species at the sn-2 position of 1-acyl LPC (18,(45)(46)(47)(48).…”

Phosphatidylcholine biosynthesis in Mitis group streptococci via host metabolite scavenging

“…A complete GPC pathway has been characterized in yeast (44, 48). It includes a dual substrate transporter, Git1, for the uptake of glycerophosphoinositol and GPC; a previously unidentified GPC acyltransferase referred to as Gpc1; and the acyltransferase Ale1 performing LPC acylation.…”

“…identified two X. campestris acyltransferases that performed the second acylation event from LPC to PC (43). Yeast possess a fully elucidated GPC pathway (44, 48).…”