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

Eur J Biochem. 2004 Sep;271(18):3752-64. doi: 10.1111/j.1432-1033.2004.04317.x.

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-[1-14C]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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / analysis
  • Acyltransferases / genetics
  • Acyltransferases / metabolism
  • Alleles
  • Amino Acid Sequence
  • Arabidopsis / enzymology*
  • Conserved Sequence
  • DNA, Complementary / genetics*
  • Escherichia coli / genetics
  • Expressed Sequence Tags
  • Gene Expression Regulation, Enzymologic
  • Lipids / analysis
  • Microsomes / enzymology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phospholipases A / metabolism*
  • Phospholipases A1
  • Phylogeny
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Sequence Homology, Amino Acid
  • Subcellular Fractions / enzymology
  • Substrate Specificity

Substances

  • DNA, Complementary
  • Lipids
  • Acyltransferases
  • Phospholipases A
  • Phospholipases A1