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J. Biol. Chem., Vol. 282, Issue 42, 30562-30569, October 19, 2007

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Identification of a Novel Lysophospholipid Acyltransferase in Saccharomyces cerevisiae^*

Shilpa Jain, NaTaza Stanford, Neha Bhagwat, Brian Seiler, Michael Costanzo, Charles Boone, and Peter Oelkers¹

From the Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104 and the Banting and Best Department of Medical Research and Department of Molecular Genetics and Microbiology, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada

The incorporation of unsaturated acyl chains into phospholipids during de novo synthesis is primarily mediated by the 1-acyl-sn-glycerol-3-phosphate acyltransferase reaction. In Saccharomyces cerevisiae, Slc1 has been shown to mediate this reaction, but distinct activity remains after its removal from the genome. To identify the enzyme that mediates the remaining activity, we performed synthetic genetic array analysis using a slc1 strain. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity, but overexpression increased activity 7-fold. Deletion of LPT1 abrogated the esterification of other lysophospholipids, and overexpression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. Although the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent K_m, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent V_max. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent V_max. Pulse-labeling of lpt1 strains showed a 30% reduction in [³H]oleate incorporation into phosphatidylcholine only. Therefore, Lpt1p, a member of the membrane-bound o-acyltransferase gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.

Received for publication, July 31, 2007 , and in revised form, August 21, 2007.

^* This work is supported in part by grants from the Canadian Institute of Health Research, Genome Canada, and Genome Ontario (to C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Drexel University, 3141 Chestnut St., Philadelphia, PA 19104. Fax: 215-895-1273; E-mail: pmo24{at}drexel.edu.

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