The long term objectives are to elucidate the molecular mechanisms that regulate the amounts and types of phospholipids required for cellular and organellar membrane synthesis, for secreted products (lipoproteins, bile, surfactant, milk fat globules) and for signal transduction processes. Physiological, biochemical, genetic and molecular biological studies conducted during the last grant period, established suitable systems for the molecular analysis of three genes encoding key phospholipid biosynthetic enzymes in S. cerevisiae, TPA1, glycerol-P acyltransferase, CPT1, DAG cholinephosphotransferase, and EPT1, DAG ethanolaminephosphotransferase. CPT1 and EPT1 were cloned, sequenced and expressed. The double null (eptl, cptl) lacked both activities and provided a suitable background for expression of chimeric CPT1-EPT1 genes. A major objective is to advance the studies from yeast to mammalian (human) cells.
The Specific Aims are: 1) To further elucidate structural and functional properties of (CPT1) and (EPT1) gene products by characterizing the protein products; by characterizing lipid substrate specificity and phospholipid cofactor requirements using individual wild type enzymes and chimeric enzymes; by determining the transmembrane topography and by investigating subcellular location; 2) To elucidate the role of the CPT1 gene, its intron, RNA and/or protein product in the pleiotropic regulation of phospholipid synthesis that occurs in yeast in response to choline and inositol; 3) To investigate the structure, function and regulation of mammalian DAG choline- and ethanolaminephosphotransferases by cloning, sequencing and expressing cDNAs encoding these enzymes from mammalian sources; 4) To understand the structure, function and regulation of yeast glycerol-P/DHAP-acyltransferase encoded by TPA1 by isolating, sequencing and expressing TPA1 in suitable systems. The enzymological studies will utilize mixed micellar assays to investigate DAG substrate specificity and phospholipid cofactor requirements of normal and chimeric enzymes. The molecular understanding of phospholipid biosynthesis in eukaryotic bells is likely to be of fundamental significance to cardiovascular disease involving lipoproteins, hypertension, stroke, and thrombosis; to alcoholism, diabetes, inflammatory disorders, digestive diseases, obesity, airway diseases including respiratory distress syndrome and asthma, and to altered signal transduction pathways involved in cancer and CNS disorders.
