Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphipathic protein that controls phosphatidylcholine synthesis.
CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) is a key regulatory enzyme in the synthesis of phosphatidylcholine in higher eukaryotes. This enzyme can interconvert between an inactive cytosolic form and an active membrane-bound form. To unravel the structure of the transferase and the mechanism of its interaction with membranes, we have cloned a cytidylyltransferase cDNA from rat liver by the oligonucleotide-directed polymerase chain reaction. Transfection of the rat clone into COS cells resulted in a 10-fold increase in cytidylyltransferase activity and content. The activity of the transfected transferase was lipid-dependent. The central portion of the derived protein sequence of the rat clone is highly homologous to the previously determined yeast cytidylyltransferase sequence [Tsukagoshi, Y., Nikawa, J. & Yamashita, S. (1987) Eur. J. Biochem. 169, 477-486]. The rat protein sequence lacks any signals for covalent lipid attachment and lacks a hydrophobic domain long enough to span a bilayer. However, it does contain a potential 58-residue amphipathic alpha-helix, encompassing three homologous 11-residue repeats. We propose that the interaction of cytidylyltransferase with membranes is mediated by this amphipathic helix lying on the surface with its axis parallel to the plane of the membrane such that its hydrophobic residues intercalate the phospholipids.
Kalmar
G B
GB
Department of Chemistry/Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
Kay
R J
RJ
Lachance
A
A
Aebersold
R
R
Cornell
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019916:Choline-Phosphate Cytidylyltransferase; D003001:Cloning, Molecular; D015870:Gene Expression; D015723:Gene Library; D008099:Liver / Q000201:enzymology*; D008297:Male; D008961:Models, Structural; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015345:Oligonucleotide Probes; D010713:Phosphatidylcholines / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D015345:Oligonucleotide Probes; D010713:Phosphatidylcholines; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase