cerulenin and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Saccharomyces cerevisiae is an ideal model eukaryote for studying fatty-acid transport. Yeast are auxotrophic for unsaturated fatty acids when grown under hypoxic conditions or when the fatty-acid synthase inhibitor cerulenin is included in the growth media. The FAT1 gene encodes a protein, Fat1p, which is required for maximal levels of fatty-acid import and has an acyl CoA synthetase activity specific for very-long-chain fatty acids suggesting this protein plays a pivotal role in fatty-acid trafficking. In the present work, we present evidence that Fat1p and the murine fatty-acid transport protein (FATP) are functional homologues. FAT1 is essential for growth under hypoxic conditions and when cerulenin was included in the culture media in the presence or absence of unsaturated fatty acids. FAT1 disruptants (fat1Delta) fail to accumulate the fluorescent long-chain fatty acid fatty-acid analogue 4, 4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-do decanoic acid (C1-BODIPY-C12), have a greatly diminished capacity to transport exogenous long-chain fatty acids, and have very long-chain acyl CoA synthetase activities that were 40% wild-type. The depression in very long-chain acyl CoA synthetase activities were not apparent in cells grown in the presence of oleate. Additionally, beta-oxidation of exogenous long-chain fatty acids is depressed to 30% wild-type levels. The reduction of beta-oxidation was correlated with a depression of intracellular oleoyl CoA levels in the fat1Delta strain following incubation of the cells with exogenous oleate. Expression of either Fat1p or murine FATP from a plasmid in a fat1Delta strain restored these phenotypic and biochemical deficiencies. Fat1p and FATP restored growth of fat1Delta cells in the presence of cerulenin and under hypoxic conditions. Furthermore, fatty-acid transport was restored and was found to be chain length specific: octanoate, a medium-chain fatty acid was transported in a Fat1p- and FATP-independent manner while the long-chain fatty acids myristate, palmitate, and oleate required either Fat1p or FATP for maximal levels of transport. Lignoceryl CoA synthetase activities were restored to wild-type levels in fat1Delta strains expressing either Fat1p or FATP. Fat1p or FATP also restored wild-type levels of beta-oxidation of exogenous long-chain fatty acids. These data show that Fat1p and FATP are functionally equivalent when expressed in yeast and play a central role in fatty-acid trafficking.

Topics: Animals; Boron Compounds; Carrier Proteins; Cell Membrane; Cerulenin; Cloning, Molecular; Coenzyme A Ligases; Fatty Acid Transport Proteins; Fatty Acids; Gene Deletion; Membrane Proteins; Membrane Transport Proteins; Mice; Oxygen; Phenotype; Saccharomyces cerevisiae; Temperature; Time Factors

The yeast Saccharomyces cerevisiae is able to utilize exogenous fatty acids for a variety of cellular processes including beta-oxidation, phospholipid biosynthesis, and protein modification. The molecular mechanisms that govern the uptake of these compounds in S. cerevisiae have not been described. We report the characterization of FAT1, a gene that encodes a putative membrane-bound long-chain fatty acid transport protein (Fat1p). Fat1p contains 623 amino acid residues that are 33% identical and 54% with similar chemical properties as compared with the fatty acid transport protein FATP described in 3T3-L1 adipocytes (Schaffer and Lodish (1994) Cell 79, 427-436), suggesting a similar function. Disruption of FAT1 results in 1) an impaired growth in YPD medium containing 25 microM cerulenin and 500 microM fatty acid (myristate (C14:0), palmitate (C16:0), or oleate (C18:1)); 2) a marked decrease in the uptake of the fluorescent long-chain fatty acid analogue boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823); 3) a reduced rate of exogenous oleate incorporation into phospholipids; and 4) a 2-3-fold decrease in the rates of oleate uptake. These data support the hypothesis that Fat1p is involved in long-chain fatty acid uptake and may represent a long-chain fatty acid transport protein.

Topics: 3T3 Cells; Adipose Tissue; Amino Acid Sequence; Animals; Boron Compounds; Carrier Proteins; Cerulenin; Fatty Acid Transport Proteins; Fatty Acids, Unsaturated; Fluorescent Dyes; Kinetics; Membrane Proteins; Membrane Transport Proteins; Mice; Microscopy, Confocal; Molecular Sequence Data; Oleic Acid; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins