AN AMPHIPATHIC ALPHA-HELIX IS THE PRINCIPLE MEMBRANE-EMBEDDED REGION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE - IDENTIFICATION OF THE 3-(TRIFLUOROMETHYL)-3-(M-[I-125]IODOPHENYL) DIAZIRINE PHOTOLABELED DOMAIN

CTP:phosphocholine cytidylyltransferase (CT), the rate controlling enz yme in phosphatidylcholine biosynthesis, is activated by reversible me mbrane binding. To investigate the membrane binding mechanism of CT, w e have used the photoreactive hydrophobic probe (trifluoromethyl)-3-(m -[I-125]iodophenyl)diazirine ([I-125]TID). Association of CT with phos phatidylcholine/oleic acid (1:1) vesicles was first demonstrated by ge l filtration analysis. Upon irradiation, CT was covalently labeled by [I-125]TID presented in phosphatidylcholine/oleic acid vesicles. This demonstrates an intercalation of part of the protein into the hydropho bic core of the membrane. To identify the membrane-embedded domain, th e chymotrypsin digestion products of [I-125]TID labeled CT were analys ed. Chymotrypsin digestion produced a set of previously defined N-term inal fragments (Craig, L., Johnson, J.E. and Cornell, R.B. (1994) J. B iol. Chem. 269, 3311), as well as several small C-terminal fragments w hich react with an anti-peptide antibody raised against the proposed a mphipathic alpha-helix. All fragments containing the amphipathic helic al region of the enzyme had [I-125]TID label associated, while the chy motryptic fragment which lacked this region was not highly labeled. Si milar fragment labeling patterns were produced when [I-125]TID was pre sented in phosphatidylcholine/oleic acid or phosphatidylcholine/diacyl glycerol vesicles, suggesting that the same domain of CT mediates bind ing to membranes containing either of the two lipid activators. A 62-r esidue synthetic peptide corresponding in sequence to the amphipathic helical region of CT was labeled with [I-125]TID, demonstrating its ab ility to intercalate independently of the rest of the protein. These r esults 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.