Mechanism and regulation of the Hsp70-based mitochondrial protein import motor

描述（由申请人提供）：所有真核生物都含有线粒体，这是一种以ATP形式产生代谢能量的复杂细胞器。线粒体还参与几个关键的细胞过程，包括氨基酸和脂质的代谢，以及铁硫簇的组装。大部分的线粒体！蛋白质在胞质核糖体上合成，并通过由跨膜蛋白传导通道和输入马达组成的内膜易位酶（TIM）复合物输入到基质中。易位由两个因素驱动：内膜的膜电位和线粒体热休克蛋白mtHsp 70（在酵母中称为Ssc 1）催化的ATP水解。刺激Ssc 1的ATP酶活性的输入马达的其他组分包括Tim 44、Pam 18和Pam 16。Tim 44协调输入马达定位到易位子的通道，促进多肽底物在跨内膜易位时的结合。Pam 18是一种特异性的J蛋白，它刺激Ssc 1的ATP酶活性。有趣的是，Pam 16与Pam 18的J结构域具有序列相似性，但不刺激Ssc 1的ATP酶活性。相反，Pam 16与Pam 18相互作用以形成有效肽导入所需的稳定复合物。这项研究旨在更好地了解这种Pam 18：Pam 16异二聚体的作用，这对细胞存活至关重要。Pam 18：Pam 16的活性构象的结构和功能将被确定，并且其对Ssc 1活性的调节将被检查以确定这种调节机制的生理意义。这些研究将采用许多不同的方法，包括酵母遗传学、细胞生物学、诱变、X射线晶体学和体外生物化学研究。Pam 18：Pam 16异二聚体与核心易位子的缔合然后将通过TIM复合物的重构来研究，这将使得许多研究由于复合物的膜结合组分的不溶性而变得困难。这些技术的发展将启动一个广泛的事业，以更好地了解蛋白质易位的内膜环境的背景下的整体机制。线粒体的正常功能对于许多细胞过程是必不可少的，包括细胞发育、钙信号传导、凋亡和衰老。因此，线粒体的功能障碍导致了许多严重的人类疾病，包括心脏病、癌症、糖尿病和神经系统疾病。因此，更好地了解线粒体蛋白质输入，这是至关重要的线粒体生物合成，是非常重要的公共卫生。

DESCRIPTION (provided by applicant): All eucaryotic organisms contain mitochondria, complex organelles that generate metabolic energy in the form of ATP. Mitochondria are also involved in several key cellular processes, including the metabolism of amino acids and lipids, and the assembly of iron-sulfur clusters. The majority of mitochondria! proteins are synthesized on cytosolic ribosomes and imported into the matrix by the translocase of the inner membrane (TIM) complex, which is composed of a transmembrane protein-conducting channel and an import motor. Translocation is driven by two factors: the electric membrane potential of the inner membrane, and ATP hydrolysis catalyzed by the mitochondrial heat-shock protein mtHsp70, termed Ssc1 in yeast. Other components of the import motor which stimulate the ATPase activity of Ssc1 include Tim44, Pam18 and Pam16. Tim44 coordinates the localization of the import motor to the channel of the translocon, facilitating the binding of polypeptide substrates as they are translocated across the inner membrane. Pam18 is a specific J protein that stimulates the ATPase activity of Ssc1. Interestingly, Pam16 has sequence similarity to the J domain of Pam18 but does not stimulate the ATPase activity of Ssc1. Instead, Pam16 interacts with Pam18 to form a stable complex which is required for efficient peptide import. This research is aimed at better understanding the role of this Pam18:Pam16 heterodimer, which is essential for cell survival. The structure and function of the active conformation of Pam18:Pam16 will be determined, and its regulation of Ssc1 activity will be examined to determine the physiological significance of such a regulatory mechanism. These studies will employ a number of different approaches, including yeast genetics, cell biology, mutagenesis, X-ray crystallography, and in vitro biochemical studies. The association of the Pam18:Pam16 heterodimer with the core translocon will then be studied by reconstitution of the TIM complex, which will enable a number of studies made difficult by the insolubility of the membrane-bound components of the complex. The development of these techniques will initiate a broad undertaking to better understand the overall mechanism of protein translocation in the context of the inner membrane environment. The proper function of mitochondria is essential to a number of cellular processes, including cell development, calcium signaling, apoptosis, and aging. Malfunctions in mitochondria are thus responsible for a number of severe human diseases including heart disease, cancer, diabetes and neurological disorders. A better understanding of mitochondrial protein import, which is critical for mitochondrial biogenesis, is therefore of great importance to public health.