The long term goal of our research is to delineate the mechanisms by which cells coordinate fatty acid transport, metabolism, and fatty acid- responsive gene regulation. These processes are complex and highly regulated. Protein-mediated long-chain fatty acid transport across the plasma membrane in higher eukaryotic cells types is relatively well established. Several distinct classes of proteins have been identified that are proposed to represent components of a membrane-bound fatty acid transport apparatus. Of these different classes, only the fatty acid transport proteins (FATP) have been identified on the basis of functional assays. This group of proteins has been identified in mouse, rat, human and yeast. In addition, on the basis of sequence homologies, there appears to be a FATP in C. elegans. In previous work our laboratory identified, cloned and characterized Saccharomyces cerevisiae FATP homologue, Fat1p encoded within FAT1. Strains which are deleted for FAT1 have been constructed and have the following phenotypes. [1] These strains fail to grow on media containing the fatty acid synthesis inhibitor cerulenin even when long chain fatty acids are supplied in the growth media; [2] They are unable to grow under anaerobic conditions on minimal media supplemented long chain unsaturated fatty acids; [3] They fail to accumulate the fluorescent long-chain fatty acid fatty acid analogue, boron dipyrromethane difluoride dodecanoic acid; and [4] These strains have a greatly diminished capacity to transport exogenous long- chain fatty acids. Each of these phenotypes are eliminated when the mutant strains are transformed with either a clone encoding the yeast Fat1p or an expression clone encoding the murine FATP. In the present work we will initiate a biochemical and molecular dissection of yeast Fat1p to determine the functional domains of this protein and to establish the kinetic properties of Fat1p-mediated fatty acid transport. We will establish which amino acids and functional domains are required for Fat1p activity and are also required for murine FATP activity. Additionally, we will investigate the link between Fat1p mediated fatty acid responsive transcriptional repression of OLE1 and we will identify additional proteins required for the fatty acid-mediated regulatory cascade.
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