Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Abstract

In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitopetagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites

“…Another eukaryotic integral membrane methyltransferase that uses AdoMet as a methyl donor is phosphatidylethanolamine N-methyltransferase (PEMT) (30). PEMT catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine through three consecutive methyl transfer reactions (31).…”

Subcellular Localization and Targeting of N-Acetylglucosaminyl Phosphatidylinositol De-N-acetylase, the Second Enzyme in the Glycosylphosphatidylinositol Biosynthetic Pathway

“…We suggest that the difference in results obtained with mouse thymoma versus HeLa cells can be attributed to the subtle, cell type-specific variation in subcellular compartmentation that is seen in many systems. For example, a similar cell type-specific variation in ER versus MAM distribution has been documented for an isoform of phosphatidylethanolamine N-methyltransferase (PEMT-2), an enzyme that is uniquely localized to MAM in rat liver subcellular fractions but is distributed uniformly between MAM and ER fractions derived from human liver (29). Other examples of proteins whose fine subcellular location depends on cell type include the ␣-mannosidases I and II (30) and the terminal glycosyltransferases ␣1,3-N-acetyl-galactosaminyltransferase (31) and ␣2,6-sialyltransferase (31, 32); these enzymes occupy different regions of the Golgi stack depending on cell type.…”

Molecular Dissection of the S-Adenosylmethionine-binding Site of Phosphatidylethanolamine N-Methyltransferase

“…The microsomes were prepared as described previously except the resultant microsomal pellet was resuspended in 75 l of Buffer X (50 mM Tris-HCl, pH 9.5, 5 mM dithiothreitol) (22). Binding assays were carried out according to a modified version of the method of Zhu et al (24).…”

“…Transfection of various plasmids encoding PEMT and mutant PEMT derivatives was performed using the FuGENE reagent as described previously (22).…”

“…Immunoblots of the recombinant PEMT protein and mutant derivatives were performed as described previously using an anti-PEMT peptide antibody at the indicated concentrations (22).…”