title: An amphipathic alpha-helix is the principle membrane-embedded region of CTP:phosphocholine cytidylyltransferase. Identification of the 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine photolabeled domain.
abstract: CTP:phosphocholine cytidylyltransferase (CT), the rate controlling enzyme in phosphatidylcholine biosynthesis, is activated by reversible membrane binding. To investigate the membrane binding mechanism of CT, we have used the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[l25I]iodophenyl)diazirine ([125I]TID). Association of CT with phosphatidylcholine/oleic acid (1:1) vesicles was first demonstrated by gel filtration analysis. Upon irradiation, CT was covalently labeled by [125I]TID presented in phosphatidylcholine/oleic acid vesicles. This demonstrates an intercalation of part of the protein into the hydrophobic core of the membrane. To identify the membrane-embedded domain, the chymotrypsin digestion products of [125I]TID labeled CT were analysed. Chymotrypsin digestion produced a set of previously defined N-terminal fragments (Craig, L., Johnson, J.E. and Cornell, R.B. (1994) J. Biol. Chem. 269, 3311), as well as several small C-terminal fragments which react with an anti-peptide antibody raised against the proposed amphipathic alpha-helix. All fragments containing the amphipathic helical region of the enzyme had [125I]TID label associated, while the chymotryptic fragment which lacked this region was not highly labeled. Similar fragment labeling patterns were produced when [125I]TID was presented in phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol vesicles, suggesting that the same domain of CT mediates binding to membranes containing either of the two lipid activators. A 62-residue synthetic peptide corresponding in sequence to the amphipathic helical region of CT was labeled with [125I]TID, demonstrating its ability to intercalate independently of the rest of the protein. These results indicate a membrane-binding mechanism for cytidylyltransferase involving the intercalation of the amphipathic alpha-helix region into the hydrophobic acyl chain core of the activating membrane.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada.
authors.forename: J E
authors.initials: JE
authors.lastname: Johnson
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
authors.forename: R B
authors.initials: RB
authors.lastname: Cornell
mesh_terms: D000818:Animals; D001389:Azirines; D002462:Cell Membrane / Q000201:enzymology*; D019916:Choline-Phosphate Cytidylyltransferase; D002918:Chymotrypsin; D003432:Cross-Linking Reagents; D004075:Diglycerides / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000737:chemistry*; D019301:Oleic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis; D010713:Phosphatidylcholines / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins; D017421:Sequence Analysis
chemical_list: D001389:Azirines; D003432:Cross-Linking Reagents; D004075:Diglycerides; D008563:Membrane Lipids; D010446:Peptide Fragments; D010455:Peptides; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D019301:Oleic Acid; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D002918:Chymotrypsin