Membrane topography of human phosphatidylethanolamine N-methyltransferase.
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 epitope-tagged, 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.
Shields
David J
DJ
Department of Biochemistry and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Lehner
Richard
R
Agellon
Luis B
LB
Vance
Dennis E
DE
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050918:Phosphatidylethanolamine N-Methyltransferase; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
D011994:Recombinant Proteins; D008780:Methyltransferases; C494489:PEMT protein, human; D050918:Phosphatidylethanolamine N-Methyltransferase