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: 6019392
• 负责人: Paul N. Black
• 金额: $ 26.45万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019392
• 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 哪些细胞协调脂肪酸的转运，代谢和脂肪酸 - 反应敏感基因调节。 这些过程很复杂且高度 受监管。 蛋白质介导的长链脂肪酸横穿 较高的真核细胞类型中的质膜相对很好 已确立的。 已经确定了几类不同类别的蛋白质 建议代表膜结合的脂肪酸的成分 运输设备。 在这些不同的类别中，只有脂肪酸 已根据 功能测定。 这组蛋白质已在小鼠中鉴定 老鼠，人类和酵母。 另外，根据序列同源性， 秀丽隐杆线虫中似乎有胖子。在以前的工作中 实验室确定，克隆和表征酿酒酵母 FATP同源物，FAT1P在FAT1中编码。 被删除的菌株 对于fat1，已经构建并具有以下表型。 [1] 这些菌株无法在含有脂肪酸合成的培养基上生长 抑制剂cerulenin即使在长链脂肪酸中也提供 增长媒体； [2]他们无法在厌氧条件下生长 最小培养基补充了长链不饱和脂肪酸； [3]他们 无法累积荧光长链脂肪酸脂肪酸 类似物，硼二吡咯烷二氟乙烯十二烷酸；和[4]这些 菌株的运输外源长大大降低 链脂肪酸。当这些表型中的每一个被消除 用编码酵母的克隆转化突变菌株 FAT1P或编码鼠FATP的表达克隆。 现在 工作我们将启动酵母的生化和分子解剖 fat1p确定该蛋白质的功能结构域，并 建立FAT1P介导的脂肪酸转运的动力学特性。 我们将确定需要哪些氨基酸和功能域 对于脂肪1p活性，也需要鼠FATP活性。 此外，我们将研究FAT1P介导的脂肪之间的联系 OLE1的酸反应转录抑制作用，我们将确定 脂肪酸介导的调节所需的其他蛋白质 级联。