Ceramides: Quantification, and regulation in the yeast Saccharomyces cerevisiae in response to hypoxia
Once thought of as primarily structural components of plasma membranes, our view of sphingolipids has expanded dramatically so that they are now appreciated as diverse and dynamic regulators of a rapidly growing number of cellular functions. A large body of evidence supports the hypothesis that many diverse stresses such as UV light heat shock DNA damage, chemotherapeutic agents, and others generate ceramide which acts as a second messenger to activate intracellular signal transduction pathways which could lead to cell-cycle arrest or apoptosis. ^ In this study, the effects of hypoxia on the regulation of ceramides in the yeast Saccharomyces cerevisiae were investigated. We developed, optimized, and compared two new methods for ceramide quantification. The first was an enzymatic assay based on the activity of purified inositolphosphoryl ceramide synthase (IPC synthase) isolated from yeast. This enzyme utilizes ceramide as a substrate and its specific activity can be related to the levels of ceramide in cells. The second method was based on normal-phase high-performance liquid chromatography (HPLC). Sphingolipids were essential for the growth of yeast cells under microaerophilic conditions (hypoxia). This was evident based on the inability of sphingolipid compensatory strains to grow under these stressful conditions. Furthermore, using the new HPLC method, we found a significant elevation in the levels of two types of ceramide: a hydroxy fatty acid-containing, and a non-hydroxy fatty acid-containing ceramide. In order to investigate the mechanisms of ceramide elevation, the expression of two enzymes involved in ceramide biosynthesis and metabolism was determined. Serine palmitoyl transferase was found to be 5.6 fold higher while IPC synthase was 25% lower in cells grown under hypoxia. Ceramide levels might have been determined by the coordinate regulation of both these enzymes. The levels of inositol-containing sphingolipids were not significantly affected by hypoxia. This strongly suggests that ceramide elevation under stressful hypoxic conditions is the result of enhanced de novo synthesis of ceramide, and not breakdown of inositol-containing complex lipids. ^
Agriculture, Food Science and Technology|Biology, Microbiology
Aida Elias Cremesti,
"Ceramides: Quantification, and regulation in the yeast Saccharomyces cerevisiae in response to hypoxia"
Dissertations and Master's Theses (Campus Access).