Genetics and Biochemistry of Fatty Acid Elongation in S. cerevisiae

Henry M Jackson Fdn For Advmt Of Military Medicine, Bethesda MD

Investigators

Abstract

ABSTRACT Proposal MCB-0078100 PI: Teresa M. Dunn This research is designed to elucidate the composition and organization of the microsomal fatty acid elongating systems. The properties and functions of cellular membranes are largely determined by the composition of their lipids, and each class of lipids has a distinct fatty acid composition. Most cellular fatty acids have 16 or 18 carbons and are synthesized by the cytosolic fatty acid synthase enzyme. However, the very long chain fatty acids (VLCFA's), with chain lengths greater than 18 carbons, are synthesized by membrane-associated enzyme systems. The function of the VLCFA's is essential to the cellular processes, since alterations in their metabolism can have devastating consequences to the cell. Three Saccharomyces cerevisiae mutants with defects in VLCFA synthesis have recently been isolated in a screen for sphingolipid synthesis mutants. The wild-type genes required for fatty acid elongation have been cloned. These cloned genes and their mutants with defective VLCFA synthesis provide the tools that are needed to resolve several important questions about the fatty acid elongating enzymes. The purpose of this study is to identify the other elongating enzymes by using a combination of genetic screens and affinity-copurification strategies. The functions of the proteins identified in these screens will be determined. Since these proteins have been conserved through evolution, the identification of the proteins in yeast will result in their identification in plants and animals as well. The cells of all living organisms such as yeast, plants, and animals, are enclosed by a thin skin of lipid and protein known as the cell membrane. The cell membrane has special openings or passages through which biochemicals enter or leave the cell. Different tissues can have different types of lipids in different proportions, and the properties and functions of the cell membranes are determined in part by the composition of their lipids. Each class of lipids has a distinct fatty acid composition. Most cellular fatty acids are made by a soluble enzyme system and contain 16 or 18 carbons. However, a special type of fatty acids (VLCFA's) has chain lengths greater than 18 carbons and is synthesized by a different enzyme system, one that is membrane-associated. A typical fatty acid of 16 or 18 carbons is lengthened by two carbon units four times in succession to give a 24 or 26 carbon length fatty acid. These VLCFA's are essential to certain cell processes since if their amounts are altered in a cell the cell will die or stop growing. These VLCFA's are usually found in a class of lipids called sphingolipids. The yeast S. cerevisiae is a good model system for studying VLCFA synthesis and function. Three yeast mutants with defects in VLCFA synthesis have recently been found. This has enabled the isolation of the corresponding genes and the proteins they encode. Normal and mutant genes can then be inserted into yeast to find out how the protein product of the gene works in making the VLCFA's. Since these proteins have been conserved through evolution, the identification of the enzymes in yeast will result in their identification in plants and animals as well. Then the function of VLCFA's and sphingolipids in higher organism can be investigated.