Identification and Characterization of the Nuclear Isoform of Drosophila melanogaster CTP:Phosphocholine Cytidylyltransferase

• Published in: Biochemistry (Easton) 2008-11, Vol.47 (45), p.11838-11846
• Main Authors: Tilley, Dana M, Evans, Chadrick R, Larson, Troy M, Edwards, Kevin A, Friesen, Jon A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cell Line; Cell Membrane - enzymology; Cell Nucleus - enzymology; Choline-Phosphate Cytidylyltransferase - chemistry; Choline-Phosphate Cytidylyltransferase - genetics; Choline-Phosphate Cytidylyltransferase - metabolism; Drosophila melanogaster; Drosophila Proteins - chemistry; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Models, Biological; Molecular Sequence Data; Sequence Homology, Amino Acid
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi801161s

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5′-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min−1 mg−1 and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 μM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg2+ > Mn2+ = Co2+ > Ca2+ = Ni2+ > Zn2+. The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.