Regulation of CTP: Phosphocholine cytidylyltransferase activity by the physical properties of lipid membranes: An important role for stored curvaturestrain energy

CTP:Phosphocholine cytidylyltransferase (CT) catalyzes the key step in phos phatidylcholine (PC) synthesis. CT is activated by binding to certain lipid membranes. The membrane binding affinity of CT can vary from micromolar to millimolar Kd, depending on the lipid composition of the target membrane. Class II CT activators like diacylglycerols and unsaturated phosphatidyleth anolamines (PE) favor inverted lipid phase formation. The mechanism(s) gove rning CT's association with class II lipid membranes and subsequent activat ion are relatively unknown. We measured CT activation by vesicles composed of PC and one of three unsaturated PEs. dioleoylglycerol (DOG), or choleste rol. For each lipid system, we estimated the stored curvature strain energy of the monolayer when confined to a relatively flat bilayer. CT binding an d activation correlate very well with the curvature strain energy of severa l chemically distinct class II lipid systems, with the exception of those c ontaining cholesterol, in which CT activation was less than the increase in curvature strain. CT activation by membranes containing DOG was reversed b y inclusion of specific lysolipids, which reduce curvature strain energy. L ysoPC, which has a larger positive curvature than lysoPE, produced greater inhibition of CT activation. Stored curvature strain energy is thus an impo rtant determinant of CT activation. Membrane interfacial polarity was inves tigated using a membrane-anchored fluorescent probe. Decreases in quenching of this interfacial probe by doxyl-PCs in class II membranes suggest the p robe adopts a more superficial membrane location. This may reflect an incre ased surface hydrophobicity of class II lipid membranes, implying a role fo r surface dehydration in CT's interactions with membranes containing class II lipids. Cholesterol, a poor activator of CT, did not affect the position ing of the polarity-sensitive probe, suggesting that one reason for its ine ffectiveness is an inability to enhance surface hydrophobicity.