Abstract
The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae. Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS). In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed. Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs. Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta). For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and [(3)H]oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry. In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants. For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable. The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters.
Highlights
The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae
For selected subsets of fat1⌬ strain transformed with YCpDB102 (FAT1) mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 M cerulenin in the presence of 100 M oleate (C18:1), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora3a,4a-diaza-S-indacene-3-dodecanoic acid and [3H]oleate, 3) levels of lignoceryl (C24:0) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry
Emerging evidence suggests that Fat1p and Faa1p work in concert and are each required for fatty acid import and targeting to specific intracellular pools and organelles [29]
Summary
DIRECTED MUTAGENESIS OF FAT1 DISTINGUISHES THE BIOCHEMICAL ACTIVITIES ASSOCIATED WITH Fat1p*. Yeast strains containing a deletion in the structural gene for Fat1p (fat1⌬) are distinct from the wild type cells on the basis of a number of growth and biochemical phenotypes [29, 33, 34] These strains 1) are compromised in their ability to grow on media containing the fatty acid synthesis inhibitor cerulenin and long-chain fatty acids; 2) show reduced uptake of radioactively labeled longchain fatty acids; 3) fail to accumulate the fluorescent longchain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4adiaza-S-indacene-3-dodecanoic acid (C1-BODIPY-C12), and 4) are defective in intracellular trafficking of exogenous fatty acids [29, 34]. Subsequent studies from our laboratory and from others have shown that Fat1p, mmFATP1, and mmFATP4 have intrinsic very long-chain fatty acyl-CoA synthetase activity in addition to playing a role in long-chain fatty acid import (29 –32) These data pose a dilemma, since very long-chain fatty acids (ϾC22) in yeast are the product of de novo synthesis and are generally not transported into the cell in response to a specific metabolic requirement. Using a combination of directed mutagenesis of the FAT1 gene and a series of biochemical studies to distinguish these two functions, we present evidence that whereas these activities are generally linked, four FAT1 mutant alleles distinguish the fatty acid transport and the very long-chain fatty acid activation functions
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