Enzymatic and cellular characterization of a catalytic fragment of CTP:phosphocholine cytidylyltransferase alpha.

To probe the mechanism of lipid activation of CTP:phosphocholine cytidylyltransferase (CCTalpha), we have characterized a catalytic fragment of the enzyme that lacks the membrane-binding segment. The kinetic properties of the purified fragment, CCTalpha236, were characterized, as well as the effects of expressing the fragment in cultured cells. CCTalpha236 was truncated after residue 236, which corresponds to the end of the highly conserved catalytic domain. The activity of purified CCTalpha236 was independent of lipids and about 50-fold higher than the activity of wild-type CCTalpha assayed in the absence of lipids, supporting a model in which the membrane-binding segment functions as an inhibitor of the catalytic domain. The kcat/Km values for CCTalpha236 were only slightly lower than those for lipid-activated CCTalpha. The importance of the membrane-binding segment in vivo was tested by expression of CCTalpha236 in CHO58 cells, a cell line that is temperature-sensitive for growth and CCTalpha activity. Expression of wild-type CCTalpha in these cells complemented the defective growth phenotype when the cells were cultured in complete or delipidated fetal bovine serum. Expression of CCTalpha236, however, did not complement the growth phenotype in the absence of serum lipids. These cells were capable of making phosphatidylcholine in the delipidated medium, so the inability of the cells to grow was not due to defective phosphatidylcholine synthesis. Supplementation of the delipidated medium with an unsaturated fatty acid allowed growth of CHO58 cells expressing CCTalpha236. These results indicate that the membrane-binding segment of CCTalpha has an important role in cellular lipid metabolism.