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Research article2004Peer reviewedOpen access

Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana

Noiriel, A; Benveniste, P; Banas, A; Stymne, S; Bouvier-Nave, P

Abstract

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-C-14]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-C-14]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degreesC and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role

Keywords

LECITHIN-CHOLESTEROL ACYLTRANSFERASE; LIPID ACYL HYDROLASE; DIACYLGLYCEROL ACYLTRANSFERASE; SACCHAROMYCES-CEREVISIAE; FATTY-ACIDS; SUBSTRATE-SPECIFICITY; OVERPRODUCES STEROLS; SIGNAL-TRANSDUCTION; TOBACCO MUTANT; A(2) ACTIVITY

Published in

European Journal of Biochemistry
2004, volume: 271, number: 18, pages: 3752-3764
Publisher: BLACKWELL PUBLISHING LTD

SLU Authors

  • Stymne, Sten

    • Department of Crop Science, Swedish University of Agricultural Sciences

  • Banas, Antoni

    • Department of Crop Science, Swedish University of Agricultural Sciences

UKÄ Subject classification

Agricultural Science

Publication identifier

  • DOI: https://doi.org/10.1111/j.1432-1033.2004.04317.x

Permanent link to this page (URI)

https://res.slu.se/id/publ/3110