The regulation and function of inositol phosphorylceramide synthase in the yeast Saccharomyces cerevisiae
IPC Synthase has been shown to be an essential membrane bound enzyme in yeast that catalyzes the transfer of PI to ceramide to yield the primary sphingolipid found in yeast, Inositolphosphorylceramide. A biochemical approach was taken to investigate the biological factors that regulate IC synthase activity in order to better understand the sphingolipid synthesis. A highly purified IPC synthase was purified over 1000-fold from the yeast Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization, as well as a series of ion exchange and hydrophobic column chromatography steps. The highly purified enzyme fraction contained three major bands at 45, 58 and 90 kDa. The 45 kDa band has been identified as the AUR1 gene that encodes for IPC synthase activity. The level of IPC synthase was shown to be regulated by the growth phase of the yeast cell. Enzyme activity and protein levels of IPC synthase were expressed at the highest levels during early and mid exponential phase. A detailed kinetic analysis of IPC synthase demonstrated that the enzyme exhibited typical saturation kinetics with respect to ceramide (Km = 0.01 mol%) and displayed allosteric regulation with respect to PI (Km = 2.5 mol% with a Hill number of 2). IPC synthase was activated by its substrate PI. Both PI and IPC synthase are regulated by inositol, a key regulator of yeast phospholipid biosynthesis. This suggests that the synthesis of yeast inositol containing sphingolipids and PI are coordinately regulated. The effect of other factors known to regulate phospholipid biosynthesis such as sphingoid bases, nucleotides and other phospholipids were also investigated. We observed that sphingoid bases inhibited IPC synthase along with CL, DAG and CDP-DAG. These studies have contributed to the understanding of how IPC synthase may be coordinately regulated with overall yeast lipid biosynthesis. IPC synthase is an important enzyme in sphingolipid biosynthesis and its activity is affected by membrane and cytosolic factors that also affect other key enzymes in the yeast phospholipid pathway. ^
Biology, Molecular|Biology, Microbiology|Chemistry, Biochemistry
Angeleah Arlene Browdy,
"The regulation and function of inositol phosphorylceramide synthase in the yeast Saccharomyces cerevisiae"
Dissertations and Master's Theses (Campus Access).