CHARACTERIZATION OF THE NONSPECIFIC LIPID TRANSFER PROTEIN-EP2 FROM CARROT (DAUCUS-CAROTA L)

The extracellular protein EP2 was previously identified as non-specifi c lipid transfer protein based on its cDNA-derived amino acid sequence . Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and ge l permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This p rotein band was recognized by the antiserum raised against a EP2-beta- galactosidase fusion-protein. Employing a fluorescent phospholipid ana log, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic ac id as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from thei r site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.