Fatty acid transport in Saccharomyces cerevisiae -: Directed mutagenesis of Fat1 distinguishes the biochemical activities associated with Fat1p
Fatty acid transport in Saccharomyces cerevisiae -: Directed mutagenesis of Fat1 distinguishes the biochemical activities associated with Fat1p
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DOI：
10.1074/jbc.m205034200
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发表时间：
2002-08-23
影响因子：
4.8
通讯作者：
Black, PN
中科院分区：
文献类型：
--
作者：
Zou, ZY;DiRusso, CC;Black, PN
脂肪酸转运蛋白 Fat1p 是酿酒酵母长链脂肪酸转运装置的一个组成部分。 Fat1p 与哺乳动物脂肪酸转运蛋白 (FATP) 和极长链酰基辅酶 A 合成酶 (VLACS) 具有显着的同源性。为了进一步了解 Fat1p 固有的功能作用（脂肪酸转运和 VLACS 活性），构建并分析了 FAT1 内一系列携带定点突变的 16 个等位基因。选择用于构建氨基酸取代的位点是基于 Fat1p 和哺乳动物 FATP 直系同源物之间的保守性，包括 ATP/AMP 和 FATP/VLACS 特征基序。将编码 Fat1p 突变形式的着丝粒和 2mu 质粒转化到含有 FAT1 缺失的酵母菌株 (fat1Delta) 中。对于选定的 FAT1 突变等位基因子集，我们观察到野生型和突变体之间的差异：1) 在 100 muM 油酸 (C-18:1) 存在下，用 45 mum 浅蓝素抑制脂肪酸合酶时的生长速率，2) 使用荧光脂肪酸的积累监测脂肪酸输入水平4,4-二氟-5-甲基-4-bora-3a,4a-二氮杂-S-indacene-3-十二烷酸和[H-3]油酸酯，3) 木蜡基 (C-24:0) CoA 合成酶活性水平，以及 4) 使用气相色谱/质谱法监测脂肪酸谱。在大多数情况下，脂肪酸/浅蓝菌素平板上的生长、脂肪酸积累水平、极长链脂肪酰辅酶A合成酶活性以及不同FAT1突变体中的脂肪酸谱之间存在相关性。对于几个值得注意的例外，脂肪酸转运和极长链脂肪酰辅酶A合成酶活性是有区别的。这些新型突变体的表征提供了一个平台，可以更全面地了解 Fat1p 在脂肪酸输入和 CoA 硫酯激活之间的联系中的作用。
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 2mu 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 mum cerulenin in the presence of 100 muM 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 [H-3]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.