CLONING AND EXPRESSION OF A NOVEL PHOSPHATIDYLETHANOLAMINE N-METHYLTRANSFERASE - A SPECIFIC BIOCHEMICAL AND CYTOLOGICAL MARKER FOR A UNIQUEMEMBRANE-FRACTION IN RAT-LIVER

Phosphatidylethanolamine N-methyltransferase catalyzes the synthesis o f phosphatidylcholine from phosphatidylethanolamine and is most active in liver. A cDNA for this enzyme from a rat liver cDNA library has be en cloned, sequenced, and expressed in COS-1 cells, McArdle-RH7777 rat hepatoma cells, and Sf9 insect cells. The expressed protein was capab le of converting phosphatidylethanolamine into phosphatidyl-choline in intact COS-1 cells, which normally have very low methyltransferase ac tivity. The calculated molecular mass of the methyltransferase protein is 22.3 kDa, which is equivalent to that of the pure protein isolated from rat liver. Comparison of the sequence of the cloned rat liver me thyltransferase with the yeast phosphatidylethanolamine methyltransfer ase PEM2 gene product revealed 44% identical amino acids and 68% simil arity in the two predicted protein sequences. A polyclonal antibody wa s raised against a synthetic peptide corresponding to the carboxyl-ter minal region of the enzyme and was affinity purified. The antibody rec ognized a single protein with a molecular mass of approximately 20 kDa when either rat liver proteins or proteins derived from the transfect ed COS-1 cells were electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate. Surprisingly, the antibody exhibited no reacti vity with endoplasmic reticulum proteins, even though the major phosph atidylethanolamine methyltransferase activity resides on this subcellu lar organelle. Instead, the antibody specifically recognized a protein in a unique subcellular membrane fraction purified from a crude mitoc hondrial preparation on a Percoll gradient. Immunocytochemical examina tion by electron microscopy showed positive labeling only in unique re gions of the hepatocytes. The data suggest that this phosphatidylethan olamine methyltransferase is a specific marker for this unique membran e fraction.