Murine FATP alleviates growth and biochemical deficiencies of yeast fat1Δ strains

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Abstract
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 (fat1Δ) 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-dodecanoic 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, β-oxidation of exogenous long-chain fatty acids is depressed to 30% wild-type levels. The reduction of β-oxidation was correlated with a depression of intracellular oleoyl CoA levels in the fat1Δ strain following incubation of the cells with exogenous oleate. Expression of either Fat1p or murine FATP from a plasmid in a fat1Δ strain restored these phenotypic and biochemical deficiencies. Fat1p and FATP restored growth of fat1Δ 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 fat1Δ strains expressing either Fat1p or FATP. Fat1p or FATP also restored wild-type levels of β-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.

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