MOLECULAR ANALYSIS OF FATTY ACID TRANSPORT IN EUKARYOTES

真核生物中脂肪酸运输的分子分析

• 批准号: 6386800
• 负责人: Paul N. Black
• 金额: $ 23万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-06 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386800
• 关键词: affinity labeling; clone cells; confocal scanning microscopy; eukaryote; fatty acid metabolism; fatty acid transport; fluorescence microscopy; fungal genetics; gene deletion mutation; gene expression; genetic regulation; genetic transcription; membrane transport proteins; molecular cloning; mutant; northern blottings; protein purification; protein structure function; site directed mutagenesis

The long term goal of our research is to delineate the mechanisms by which cells coordinate fatty acid transport, metabolism, and fatty acid- responsive gene regulation. These processes are complex and highly regulated. Protein-mediated long-chain fatty acid transport across the plasma membrane in higher eukaryotic cells types is relatively well established. Several distinct classes of proteins have been identified that are proposed to represent components of a membrane-bound fatty acid transport apparatus. Of these different classes, only the fatty acid transport proteins (FATP) have been identified on the basis of functional assays. This group of proteins has been identified in mouse, rat, human and yeast. In addition, on the basis of sequence homologies, there appears to be a FATP in C. elegans. In previous work our laboratory identified, cloned and characterized Saccharomyces cerevisiae FATP homologue, Fat1p encoded within FAT1. Strains which are deleted for FAT1 have been constructed and have the following phenotypes. [1] These strains fail to grow on media containing the fatty acid synthesis inhibitor cerulenin even when long chain fatty acids are supplied in the growth media; [2] They are unable to grow under anaerobic conditions on minimal media supplemented long chain unsaturated fatty acids; [3] They fail to accumulate the fluorescent long-chain fatty acid fatty acid analogue, boron dipyrromethane difluoride dodecanoic acid; and [4] These strains have a greatly diminished capacity to transport exogenous long- chain fatty acids. Each of these phenotypes are eliminated when the mutant strains are transformed with either a clone encoding the yeast Fat1p or an expression clone encoding the murine FATP. In the present work we will initiate a biochemical and molecular dissection of yeast Fat1p to determine the functional domains of this protein and to establish the kinetic properties of Fat1p-mediated fatty acid transport. We will establish which amino acids and functional domains are required for Fat1p activity and are also required for murine FATP activity. Additionally, we will investigate the link between Fat1p mediated fatty acid responsive transcriptional repression of OLE1 and we will identify additional proteins required for the fatty acid-mediated regulatory cascade.

我们研究的长期目标是通过以下方式描述机制： 哪些细胞协调脂肪酸运输、代谢和脂肪酸- 响应性基因调控。 这些过程复杂且高度 受监管。 蛋白质介导的长链脂肪酸转运 高等真核细胞类型的质膜相对较好 已确立的。 已鉴定出几种不同类别的蛋白质 被提议代表膜结合脂肪酸的成分 运输器具。 在这些不同的类别中，只有脂肪酸 转运蛋白（FATP）的鉴定基于 功能测定。 这组蛋白质已在小鼠体内得到鉴定， 大鼠、人类和酵母。 此外，根据序列同源性， 线虫中似乎存在 FATP。在之前的工作中我们的 实验室鉴定、克隆和鉴定酿酒酵母 FATP 同源物，Fat1p 在 FAT1 内编码。 被删除的菌株 已构建 FAT1 并具有以下表型。 [1] 这些菌株无法在含有脂肪酸合成的培养基上生长 即使在提供长链脂肪酸时，也能抑制淡蓝素 生长培养基； [2] 它们无法在厌氧条件下生长 补充长链不饱和脂肪酸的基本培养基； [3] 他们 未能积累荧光长链脂肪酸 类似物，二氟化硼十二烷酸；和 [4] 这些 菌株运输外源长链的能力大大减弱 链脂肪酸。当这些表型中的每一个都被消除时 突变菌株用编码酵母的克隆转化 Fat1p 或编码鼠 FATP 的表达克隆。 在现在 我们将启动酵母的生化和分子解剖工作 Fat1p 确定该蛋白质的功能域并 建立 Fat1p 介导的脂肪酸运输的动力学特性。 我们将确定需要哪些氨基酸和功能域 Fat1p 活性，也是小鼠 FATP 活性所必需的。 此外，我们将研究 Fat1p 介导的脂肪之间的联系 OLE1 的酸反应性转录抑制，我们将鉴定 脂肪酸介导的调节所需的额外蛋白质 级联。