Abstract
The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were over-expressed 20-30-fold in COS cells. Most of the CT activities were found in the cytosolic fraction. The regulation of the yeast CT activity (Y-CT) by lipids was characterized for the first time in comparison with the regulation of the well-studied rat CT (R-CT). Sonicated vesicles composed of egg phosphatidylcholine (PC) or 1-stearoyl-2-oleoyl PC had no effect on Y-CT and only slightly stimulated R-CT activity. Both CTs were activated 10-50-fold by the anionic lipids cardiolipin, phosphatidyl-glycerol, phosphatidylinositol and oleic acid. The effects of varying the vesicle concentration and the mol% of anionic lipid in PC vesicles were tested. The concentration optima for the activation of Y-CT by oleic acid or anionic phospholipids were 5-10-fold lower than those for R-CT. For example, the stimulation of Y-CT activity by phosphatidylglycerol vesicles was optimal between 5 and 15 microM and declined at higher concentrations, but R-CT activation by these vesicles saturated at approximately 25 microM. The positively charged aminolipid sphingosine antagonized the stimulation by oleic acid of both Y-CT and R-CT. Y-CT activity was insensitive to PC vesicles containing the neutral lipids diacylglycerol, monoacylglycerol or oleyl alcohol. However, R-CT was stimulated 10-20-fold by vesicles containing these neutral lipids. Translocation of the CTs to microsomal membranes enriched with anionic or neutral lipids was compared. Oleic acid enrichment promoted translocation of Y-CT and R-CT, whereas diacylglycerol promoted only R-CT translocation. These data show that the activity of Y-CT is lipid-sensitive. Y-CT is affected only by charged lipids, whereas R-CT responds to charged and neutral lipid activators. The data are consistent with different modes of interaction of the two CTs with lipids.