PROTEIN IMPORT INTO MITOCHONDRIA

蛋白质导入线粒体

• 批准号: 6555838
• 负责人: DEBKUMAR PAIN
• 金额: $ 18.55万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6555838
• 关键词: chimeric proteins; glutathione transferase; green fluorescent proteins; guanine nucleotide binding protein; laboratory rabbit; membrane transport proteins; mitochondrial membrane; molecular cloning; protein signal sequence; protein transport; yeast two hybrid system

The goal of this proposal is to define the mechanism by which GTP participates in mitochondrial protein import using yeast as a model system. We have demonstrated for first time that GTP hydrolysis plays a direct and essential role during import of urea-denatured precursors into the matrix. Import of these denatured precursors does not depend on ATP-dependent interactions with cytosolic chaperones. Both external GTP and matrix ATP are required for efficient import. Protein import into or across mitochondrial outer and inner membranes is a multi-step process. GTP may regulate recognition and subsequent unfolding of precursors at the mitochondrial surface and/or provide energy for transmembrane movement of precursors into the organelle. The first objective is to define the exact steps that require GTP. The GTP effect is likely mediated by GTP-binding proteins. Thus far, a GTP-binding protein has not been identified as part of the outer (Tom) or inner membrane (Tim) translocases. The second objective is to identify these critical GTP-binding proteins. A chimeric protein (pPHPrA) has been constructed, comprising an authentic N-terminal mitochondrial precursor (delta-1 pyrroline-5-carboxylate dehydrogenase) lined, through glutathione S-transferase, to IgG binding domains derived from staphylococcal Protein A. The native pPGPrA becomes irreversibly arrested, both in vitro and in vivo, while being translocated across the outer and inner membranes of mitochondria. During in vivo import of pPGPrA, the outer and inner membranes of mitochondria become progressively "zippered" together, forming long stretches of close contact. Affinity chromatography of solubilized ~zippered~ membranes has identified several core components of both translocation channels. These proteins are specifically associated with the arrested PGPrA intermediates. They include three GTP- binding proteins, as well as all know essential elements of Tom and Tim. The final goal is to determine how the newly identified GTP- binding proteins participate (together with other Toms and Tims) in distinct stages during mitochondrial protein import. This project is of direct health-related significance, since perturbation of mitochondrial function is associated with specific mitochondrial disease, aging, and certain neurodegenerative disorders. There is strong evidence that the constituents of mitochondrial translocases and their role in protein import are conserved between yeast and humans.

该提议的目的是定义GTP的机制 参与使用酵母作为模型的线粒体蛋白进口 系统。 我们首次证明了GTP水解 在进口尿素降级期间扮演直接和重要的角色 前体进入矩阵。 这些变性前体的导入 不依赖于ATP依赖性与胞质的相互作用 伴侣。 外部GTP和矩阵ATP都需要 有效的导入。 蛋白质进口或跨线粒体外部 内部膜是一个多步骤过程。 GTP可能会调节 识别和随后在前体展开 线粒体表面和/或提供跨膜的能量 前体向细胞器运动。 第一个目标是 定义需要GTP的确切步骤。 GTP效应可能是 由GTP结合蛋白介导。 到目前为止，GTP结合蛋白 尚未被识别为外膜或内膜的一部分 （蒂姆）易位酶。 第二个目标是确定这些 关键的GTP结合蛋白。 嵌合蛋白（PPHPRA）已经 构造，包括真实的N末端线粒体 前体（Delta-1吡罗琳-5-羧酸甲酯脱氢酶），衬有 通过谷胱甘肽S-转移酶，到IgG结合域得出 从葡萄球菌蛋白A中 在体外和体内不可逆转地逮捕 跨线粒体的外膜和内膜易位。 在体内进口PPGPRA期间，外膜的外膜 线粒体逐渐“拉链”在一起，形成很长 紧密接触。 溶解的亲和力色谱法 〜拉链〜膜已经确定了两者的几个核心组件 易位通道。 这些蛋白质特别相关 与被捕的PGPRA中间体一起。 它们包括三个GTP- 结合蛋白质以及所有人都知道汤姆的基本要素 蒂姆。 最终目标是确定新确定的GTP-如何 结合蛋白（与其他TOM和TIMS一起）参加 线粒体蛋白进口期间的不同阶段。 该项目具有直接与健康相关的意义，因为 线粒体功能的扰动与特定相关 线粒体疾病，衰老和某些神经退行性 疾病。 有强有力的证据表明 线粒体易位酶及其在蛋白质进口中的作用是 酵母和人之间的保守。