Abstract
BackgroundPhosphatidic acid (PA) is a key regulated intermediate and precursor for de novo biosynthesis of all glycerophospholipids. PA can be synthesized through the acylation of lysophosphatidic acid (LPA) by 1-acyl-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase, LPAAT). Recent findings have substantiated the essential roles of acyltransferases in various biological functions.Methodologies/Principal FindingsWe used a flow-injection-based lipidomic approach with ∼200 multiple reaction monitoring (MRM) transitions to pre-screen fatty acyl composition of phospholipids in the yeast Saccharomyces cerevisiae mutants. Dramatic changes were observed in fatty acyl composition in some yeast mutants including Slc1p, a well-characterized LPAAT, and Cst26p, a recently characterized phosphatidylinositol stearoyl incorporating 1 protein and putative LPAAT in S. cerevisiae. A comprehensive high-performance liquid chromatography–based multi-stage MRM approach (more than 500 MRM transitions) was developed and further applied to quantify individual phospholipids in both strains to confirm these changes. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants. These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates.ConclusionsWe found that Slc1p utilized fatty acid (FA) 18:1 and FA 14:0 as substrates to synthesize corresponding PAs; moreover, it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0) in S. cerevisiae. We also identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant. Our current findings expand the enzymes and existing scope of acyl-CoA donors for glycerophospholipid biosynthesis.
Highlights
Glycerophospholipids are major components of cellular membranes and are involved in a wide range of biological processes[1]
We found that Slc1p utilized fatty acid (FA) 18:1 and FA 14:0 as substrates to synthesize corresponding Phosphatidic acid (PA); it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0) in S. cerevisiae
We identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant
Summary
Glycerophospholipids are major components of cellular membranes and are involved in a wide range of biological processes[1]. Acyltransferases including glycerol-3-phosphate acyltransferase (GPAT), 1-acyl-3-phosphate acyltransferase (AGPAT) ( called lysophosphatidic acid acyltransferase, LPAAT), LPLAT (lysophospholipid acyltransferase) and diacylglyceride acyltransferase families play essential roles in acyl substrate selection for deacylation and reacylation reactions (Lands cycle) during glycerophospholipid biosynthesis [2]. Recent findings have further substantiated the essential roles of acyltransferases in membrane diversity, curvature, and asymmetric distribution of fatty acyls (FA) within individual phospholipids[4,5,6]. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates
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