14] Inositolphosphoryl ceramide synthase from yeast

Abstract

Publisher Summary Inositolphosphoryl ceramide (IPC) synthase catalyzes the transfer of inositol phosphate from phosphatidylinositol (PI) to ceramide forming diacylglycerol and IPC, a major yeast sphingolipid and precursor of the yeast sphingolipids, mannosylinositolphosphoryl ceramide and mannosyldiinositolphosphoryl ceramide. The enzyme, encoded by the yeast AUR1 gene, and its product, IPC, are essential to the growth and viability of Saccharornyces cerevisiae. IPC synthase activity is associated with the microsomal membrane fraction of yeast, and the expression of IPC synthase activity is regulated by inositol and the growth phase of S. cerevisiae . Methods to measure IPC synthase activity from yeast microsomal membranes and Triton X-100-solubilized yeast microsomes have been reported. This chapter describes the purification and properties of the enzyme. The overall purification of IPC synthase over the cell extract is 3425-fold with an activity yield of 23%. Examination of the purified enzyme by sodium dodecyl sulfate gel electrophoresis reveals four major protein bands, suggesting that the enzyme is highly purified but not to homogeneity. The four major protein bands have minimum subunit molecular weights of 97,000, 66,000, 50,000, and 46,000.