Sphingolipids are widespread in nature but their functions are poorly understood. Fungi contain sphingolipids that are distinguished from animal sphingolipids by the presence of phosphoinositol. The pathway of sphingolipid biosynthesis in yeast is schematically, precursors---long chain base ceramide-- sphingolipids. Saccharomyces cerevisiae contains a small number of sphingolipids and offers a unique opportunity to use molecular genetic techniques in conjunction with biochemical techniques to study sphingolipids. As a result of our efforts yeast are the only eucaryote for which there are know mutants defective in long chain base synthesis and for which a gene in the biosynthetic pathway has been isolated. Mutants in later steps of sphingolipid biosynthesis have not been isolated in any eucaryote; however we propose their isolation by a novel enrichment procedure we have developed. To understand the details of the sphingolipid biosynthetic pathway, its regulation, and the function of these lipids in S. cerevisiae, we will characterize the genes responsible for long chain base synthesis by use of long chain base auxotrophs (Lcb-) already isolated as well as additional ones obtained by an effective selection method; one of these genes has already been cloned (LCB1). We propose to isolate genes later in this pathway by use of temperature sensitive mutants obtained by a similar selection method. Genes that direct sphingolipid synthesis will be cloned and we will determine their transcript size, direction of transcription, transcription start sites, and nucleotide sequence. The regulation of transcription of these genes will be examined. The functions of yeast sphingolipids will be probed by isolating and characterizing second-site suppressor genes and by overexpression of the cloned sphingolipid biosynthesis genes. Yeast sphingolipids provide a local focus for the rational design of antifungal agents for use in alleviating human fungal infections.
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