Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIα at the trans-Golgi network

Type II phosphatidylinositol 4-kinase II alpha (PI4KII alpha) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KII alpha has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KII alpha mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KII alpha activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KII alpha to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KII alpha demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KII alpha was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KII alpha. Further measurements revealed that the reduction in the mobile fraction of PI4KII alpha correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.-Minogue, S., K. M. E. Chu, E. J. Westover, D. F. Covey, J. J. Hsuan, and M. G. Waugh. Relationship among phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KII alpha at the trans Golgi network. J. Lipid Res. 2010. 51: 2314-2324.