Cytidylyltransferase-binding protein is identical to transcytosis-associated protein (TAP/p115) and enhances the lipid activation of cytidylyltransferase.
We previously identified a protein from rat liver that binds CTP:phosphocholine cytidylyltransferase (CT). We have now purified this protein (cytidylyltransferase-binding protein (CTBP)) from rat liver. The purification involved precipitation at pH 5 and extraction of the precipitate with buffer, followed by sequential chromatography on DEAE-Sepharose and butyl-agarose. Final purification was accomplished by either preparative electrophoresis or hydroxylapatite chromatography. Amino acid sequences from six peptides derived from pure CTBP matched sequences in transcytosis-associated protein (TAP) with 98% identity. Thus, CTBP was positively identified to be TAP. Purified CTBP increased the activity of purified CT measured with phosphatidylcholine (PC)/oleic acid. In the absence of PC/oleic acid, CTBP did not stimulate CT activity. Dilution of CT to reduce the Triton X-100 concentration produced a loss of CT activity. The lost activity was recovered by the addition of CTBP plus PC/oleic acid to the assay, but not by the addition of either PC/oleic acid or CTBP alone. Removal of CTBP from purified preparations by immunoprecipitation with CTBP antibodies eliminated the activation of CT. Both CT and CTBP were shown to bind to PC/oleic acid liposomes. The formation of complexes between CT and CTBP in the absence of PC/oleic acid liposomes could not be demonstrated. These results suggest that CTBP functions to modify the interaction of CT with PC/oleic acid liposomes, resulting in an increase in the catalytic activity perhaps by the formation of a ternary complex between CT, CTBP, and lipid. Overall, these results suggest that CTBP (TAP) may function to coordinate the biosynthesis of phosphatidylcholine with vesicle transport.
Feldman
D A
DA
Veterans Administration Medical Center and the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48105, USA.
Weinhold
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D000077614:Golgi Matrix Proteins; D050356:Lipid Metabolism; D008081:Liposomes; D008099:Liver / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D000077614:Golgi Matrix Proteins; D008081:Liposomes; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C091580:vesicular transport factor p115; D009713:Nucleotidyltransferases