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|Title:||Characterization of substrate preference for Slc1p and Cst26p in saccharomyces cerevisiae using lipidomic approaches and an LPAAT activity assay||Authors:||Shui, G.
|Issue Date:||2010||Citation:||Shui, G., Guan, X.L., Gopalakrishnan, P., Xue, Y., Goh, J.S.Y., Yang, H., Wenk, M.R. (2010). Characterization of substrate preference for Slc1p and Cst26p in saccharomyces cerevisiae using lipidomic approaches and an LPAAT activity assay. PLoS ONE 5 (8) : -. ScholarBank@NUS Repository. https://doi.org/10.1371/journal.pone.0011956||Abstract:||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 wellcharacterized 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. © 2010 Shui et al.||Source Title:||PLoS ONE||URI:||http://scholarbank.nus.edu.sg/handle/10635/108296||ISSN:||19326203||DOI:||10.1371/journal.pone.0011956|
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