Molecular Genetic Analysis of Yeast Sphingolipids

Sphingolipids are emerging as key elements in membrane organization and function and as regulators of signal transduction pathways that mediate stress protection and control growth. Many of these roles seem to be conserved between yeast and mammals. Progress has been made in understanding the functions of sphingolipids in membranes and in signaling but our knowledge remains rudimentary and much effort will be required to achieve a more thorough understanding. Our previous studies demonstrated that sphingolipids are essential for growth of Saccharomyces cerevisiae cells and that they play roles in heat stress. Three Specific Aims will build upon these studies. The first aim is to determine why sphingolipids are necessary for exocytosis during heat stress. Specifically, to determine which sphingolipid is necessary, which step requires sphingolipids, and what the function of the required sphingolipid is. The second aim is to characterize putative protein kinases regulated by sphingoid long chain bases. The protein kinases are Pkh 1p and Pkh2p, homologs of mammalian phosphoinositide-dependent kinases (PDKs), and Yck2p, a homolog of mammalian casein kinases. These proteins regulate poorly characterized signaling pathways necessary for yeast cell growth and stress protection. In addition, the yeast proteome will be screened, using a newly developed protein chip technique, for proteins that bind specific sphingolipids in order to identify novel signaling pathways and cellular processes that require sphingolipids for function. The third aim is to determine why the very long chain-fatty acid (VLCFA, C26) component of sphingolipids is necessary for acidification of vacuoles (lysosomes) by the vacuolar ATPase. VLCFAs are present in all eucaryotes but their functions remain unknown. These studies will reveal new roles for sphingolipids in membrane function, such as in lipid rafts, and in signaling pathways that are crucial for stress protection, growth control and essential physiological processes. The results of the proposed studies should aid in understanding mammalian signaling pathways and help to reveal useful targets for pharmacological intervention in human ailments including cancer, atherosclerosis and neurological disorders.