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Journal of Lipid Research, Vol. 49, 1794-1806, August 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae

Kjell Stålberg^*, Andrea C. Neal^*, Hans Ronne and Ulf Ståhl^1,

^* Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden
Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, Alnarp, Sweden

This work was supported by two Swedish University of Agricultural Sciences strategic research programs, The Biological Factory and AgriFunGen.

Published, JLR Papers in Press, April 22, 2008.

¹ To whom correspondence should be addressed. e-mail: ulf.stahl{at}vbsg.slu.se

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acyl-CoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V_max of 8.7 nmol/min/mg protein and an apparent K_m of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn²⁺ ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [³H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked. This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.

Supplementary key words glycerophosphocholine acyltransferase • membrane • recycling • remodeling • fatty acid • phospholipid • phosphatidylethanolamine • lysophosphatidylcholine • glycerophosphoethanolamine • Saccharomyces cerevisiae

Abbreviations: DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; GPC, glycerophosphocholine; GPCAT, glycerophosphocholine acyltransferase; GPE, glycerophosphoethanolamine; GPG, glycerophosphoglycerol; GPI, glycerophosphoinositol; GPS, glycerophosphoserine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPLAT, lysophospholipid acyltransferase; NEM, N-ethylmaleimide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; WT, wild type

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