Fatty acid transport in Saccharomyces cerevisiae

Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p

Zhiying Zou, Concetta C DiRusso, Vlasta Ctrnacta, Paul N Black

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83 Citations (Scopus)

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 2μ plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Δ). 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 μM cerulenin in the presence of 100 μM oleate (C18:1), 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 [3H]oleate, 3) levels of lignoceryl (C24:0) 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.

Original languageEnglish (US)
Pages (from-to)31062-31071
Number of pages10
JournalJournal of Biological Chemistry
Volume277
Issue number34
DOIs
StatePublished - Aug 23 2002

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Mutagenesis
Yeast
Saccharomyces cerevisiae
Fatty Acids
Fatty Acid Transport Proteins
Cerulenin
Coenzyme A Ligases
Acyl Coenzyme A
lauric acid
Coenzyme A
Oleic Acid
Yeasts
Alleles
Fatty Acid Synthases
Amino Acid Substitution
Adenosine Monophosphate
Ligases
Growth
Gas Chromatography-Mass Spectrometry
Gas chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Fatty acid transport in Saccharomyces cerevisiae: Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p",
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 2μ plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Δ). 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 μM cerulenin in the presence of 100 μM oleate (C18:1), 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 [3H]oleate, 3) levels of lignoceryl (C24:0) 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.",
author = "Zhiying Zou and DiRusso, {Concetta C} and Vlasta Ctrnacta and Black, {Paul N}",
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T2 - Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p

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AU - Black, Paul N

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