Abnormalities in lipid metabolism are major contributing factors to various disease states including obesity, diabetes, and cardiomyopathies. Together, these diseases are the leading cause of death in the United States and most other developed countries. It is hypothesized that high circulating levels of lipids lead to excessive intraceliular accumulation of fatty acids and the resultant lipoatrophies of cells and tissues contributing to these disease states. Prevention and therapeutic intervention are therefore desirable and will require an understanding of the underlying molecular mechanisms leading to aberrant import and trafficking of fatty acids. Therefore, the focus of the present work is to determine the components and mechanisms governing fatty acid transport across a biological membrane. Current models suggest fatty acid import occurs by a mechanism involving simple diffusion and protein mediation. The present proposal employs Saccharomyces cerevisiae as a model system to define the transport mechanism. In S. cerevisiae long chain fatty acid import requires the putative transport protein Fat1p, a member of the highly conserved FATP family of proteins, and fatty acyl CoA synthetase (Faa1p or Faa4p). The FATP family shares amino acid identities with the very long-chain acyl CoA synthetase enzyme family. Current evidence supports the conclusion the protein has two activities: one in fatty acid transport and the other in activation of very long-chain fatty acids. Previous work from our laboratory demonstrated Fat1p and the murine homologue, mmFATP1, are functional orthologues. Fat1p is required when yeast are grown under anaerobic conditions to facilitate import of unsaturated fatty acids and is required for growth on media containing long chain fatty acids when fatty acid synthase is inhibited chemically. Growth and biochemical deficiencies resulting from mutations in the gene encoding Fat1p are alleviated by expression of mmFATP1 in yeast. Additional studies using directed mutagenesis of FAT1 have defined, through analyses of altered Fat1p proteins, functional domains and distinguished transport of long chain fatty acids from activation of very long chain fatty acids. The goals of the current experimental plan are to: [I] Define substrate specificity of different isoforms of mammalian FATP (mmFATP1, 2, 3, 4, and 5) expressed in S. cerevisiae for fatty acid transport and activation. [II] Further delimit subdomains and specific amino acid residues within Fat1p, which are essential for fatty acid transport, very long-chain fatty acyl CoA synthetase activity or both. [Ill] Define biochemical role of Fat1p in the trafficking of exogenous long-chain fatty acids using plasma membrane vesicles. ? ?
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