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

描述(申请人提供)：所有真核生物都含有线粒体，这是一种复杂的细胞器，以三磷酸腺苷的形式产生代谢能量。线粒体还参与了几个关键的细胞过程，包括氨基酸和脂类的新陈代谢，以及铁-硫簇的组装。线粒体的大部分！蛋白质在胞质核糖体上合成，并通过内膜(TIM)复合体的易位酶输入到基质中，内膜复合体由跨膜蛋白传导通道和输入马达组成。转位是由两个因素驱动的：内膜的电膜电位和线粒体热休克蛋白mtHsp70催化的ATP水解酶，在酵母中称为Ssc1。其他刺激SSC1 ATPase活性的进口马达成分包括TIM44、PAM18和PAM16。Tim44协调输入马达到转运子通道的定位，促进多肽底物跨内膜转运时的结合。Pam18是一种特异性J蛋白，能刺激Ssc1的ATPase活性。有趣的是，Pam16与Pam18的J区序列相似，但不能刺激Ssc1的ATPase活性。相反，Pam16与Pam18相互作用形成稳定的复合体，这是有效的多肽导入所必需的。这项研究旨在更好地了解Pam18：Pam16异源二聚体的作用，它是细胞生存所必需的。将确定Pam18：Pam16活性构象的结构和功能，并将检测其对Ssc1活性的调节，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射线结晶学和体外生化研究。然后，将通过TIM复合体的重构来研究Pam18：Pam16异二聚体与核心转位子的关联，这将使一些因复合体的膜结合成分的不溶性而变得困难的研究成为可能。这些技术的发展将启动一项广泛的工作，以更好地了解内膜环境中蛋白质转运的整体机制。线粒体的正常功能对许多细胞过程是必不可少的，包括细胞发育、钙信号、细胞凋亡和衰老。因此，线粒体的故障导致了许多严重的人类疾病，包括心脏病、癌症、糖尿病和神经疾病。因此，更好地了解线粒体蛋白质输入对线粒体生物发生至关重要，对公众健康具有重要意义。

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.