Translocation of CTP: phosphocholine cytidylyltransferase from cytosol to membranes in HeLa cells: stimulation by fatty acid, fatty alcohol, mono- and diacylglycerol.
Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP: phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, 80-90% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyltransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCl. These results suggest that a variety of compounds with differing head group size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.
D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D019916:Choline-Phosphate Cytidylyltransferase; D003600:Cytosol / Q000201:enzymology; D005233:Fatty Alcohols / Q000494:pharmacology*; D005989:Glycerides / Q000494:pharmacology*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000494:pharmacology*; D010743:Phospholipids / Q000494:pharmacology
D005233:Fatty Alcohols; D005989:Glycerides; D009829:Oleic Acids; D010169:Palmitic Acids; D010743:Phospholipids; C010268:oleyl alcohol; D019301:Oleic Acid; D019308:Palmitic Acid; C005953:monoolein; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase