title: Identification of an inhibitory domain of CTP:phosphocholine cytidylyltransferase.
abstract: The function of the putative amphipathic helices between residues 236 and 314 of CTP:phosphocholine cytidylyltransferase was examined by constructing two truncation mutants; CT314 was missing the entire phosphorylation segment, whereas CT236 was missing both the region with the putative amphipathic helices and the phosphorylation segment. Stable cells lines expressing these truncation mutants in Chinese hamster ovary 58 cells were isolated and characterized. CT314 was predominantly soluble in control cells but became membrane-associated in cells treated with oleate, which also causes translocation of wild-type cytidylyltransferase. CT236 was found to be soluble both in control cells and in cells treated to cause translocation. These results strongly suggest that the membrane-binding site is located within residues 237-314. When assayed for activity in vitro, the mutant forms were catalytically active in the presence of exogenous lipids. CT236, moreover, was as active in the absence of lipids as in their presence, whereas CT314 required lipids for activity. The rate of phosphatidylcholine synthesis in cells expressing CT236 was considerably higher than in wild-type cells, consistent with the enzyme being constitutively active in the cells. These results indicate that residues 237-314 constitute an inhibitory segment; when this segment is removed from the catalytic domain by truncation or by binding to membranes, an inhibitory constraint is removed and cytidylyltransferase is activated.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor 48109-0606, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Kent
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019916:Choline-Phosphate Cytidylyltransferase; D006224:Cricetinae; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D013329:Structure-Activity Relationship
chemical_list: D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase