Characterization of Substrate Preference for Slc1p and Cst26p in <i>Saccharomyces cerevisiae</i> Using Lipidomic Approaches and an LPAAT Activity Assay

Background Phosphatidic 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 Findings We 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. Conclusions We 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.研究背景 磷脂酸（Phosphatidic acid, PA）是所有甘油磷脂从头生物合成的关键调控中间体与前体物质。磷脂酸可通过1-酰基-3-磷酸酰基转移酶（亦称为溶血磷脂酸酰基转移酶，LPAAT）催化溶血磷脂酸（lysophosphatidic acid, LPA）的酰化反应合成。近期研究证实了酰基转移酶在多种生物学功能中的核心作用。 方法学与主要研究结果 本研究采用基于流动注射的脂质组学方法，结合约200个多反应监测（MRM）通道，对酿酒酵母（Saccharomyces cerevisiae）突变体中磷脂的脂肪酰基组成进行预筛选。在部分酿酒酵母突变体中观察到脂肪酰基组成发生显著变化，其中包括功能已明确的LPAAT——Slc1p，以及近期在酿酒酵母中鉴定的磷脂酰肌醇硬脂酰掺入1蛋白（Cst26p），其被推测为潜在的LPAAT。本研究开发了一套基于高效液相色谱的多阶段MRM分析方法（包含500余个MRM通道），并进一步用于定量两种菌株中的单个磷脂组分，以验证上述变化。本研究数据揭示了潜在的脂肪酰基底物，以及可弥补Cst26p与Slc1p突变体缺陷的脂肪酰基种类。上述结果与采用C17-LPA与酰基辅酶A作为底物的非放射性LPAAT酶活实验结果一致。 研究结论 本研究发现Slc1p以脂肪酸（fatty acid, FA）18:1与FA 14:0为底物合成对应的磷脂酸；此外，Slc1p可能是酿酒酵母中唯一负责催化饱和短链脂肪酰基（12:0与10:0）进行酰化反应的酰基转移酶。本研究同时确定FA 18:0、FA 16:0、FA 14:0与外源性FA 17:0是Cst26p的优选底物，因为通过转入带有绿色荧光蛋白（GFP）标签的CST26基因可恢复CST26突变体的磷脂谱特征。本研究结果拓展了甘油磷脂生物合成中所涉及的酶类及现有酰基辅酶A供体的范围。