Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.
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-[1-14C]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-[1-14C]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 degrees C 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.
Noiriel
Alexandre
A
Institut de Biologie Moléculaire des Plantes du CNRS, Département Isoprénoïdes, Institut de Botanique, Strasbourg, France.
Benveniste
Pierre
P
Banas
Antoni
A
Stymne
Sten
S
Bouvier-Navé
Pierrette
P
D000217:Acyltransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D015971:Gene Expression Regulation, Enzymologic; D008055:Lipids / Q000032:analysis; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000378:metabolism*; D054466:Phospholipases A1; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D018076:DNA, Complementary; D008055:Lipids; D000217:Acyltransferases; D010741:Phospholipases A; D054466:Phospholipases A1