tailieunhanh - Báo cáo khoa học: Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana

During a search for cDNAs encoding plant sterol acyl-transferases, we isolated four full-length cDNAs fromAra-bidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expressionof one of these cDNAs,AtLCAT3 (At3g03310), invarious 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 lysophospho-lipids and free fatty acids increased | Eur. J. Biochem. 271 3752-3764 2004 FEBS 2004 doi Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana Alexandre Noiriel1 Pierre Benveniste1 Antoni Banas2 Sten Stymne2 and Pierrette Bouvier-Nave1 1Institut de Biologie Moléculaire des Plantes du CNRS Departement Isoprénoides Institut de Botanique Strasbourg France department of Crop Science Swedish University of Agricultural Sciences Alnarp Sweden During a search for cDNAs encoding plant sterol acyltransferases we isolated four full-length cDNAs from Ara-bidopsis 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 lysophospho-lipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di- 1-14C oleyl phosphatidylcholine both the lysophospho-lipid 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- 1-14C acyl phosphatidylcholine the distribution of the labelling between the free fatty acid and the lysophospha-tidylcholine 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. Lysophospatidylcho-line was a .