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。线粒体还参与了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。大多数线粒体！蛋白质在细胞质核糖体上合成，并通过由跨膜蛋白质传导通道和输入马达组成的内膜转位酶（TIM）复合物导入基质。易位由两个因素驱动：内膜的膜电位和线粒体热休克蛋白mtHsp70（酵母中称为Ssc1）催化的ATP水解。刺激Ssc1 atp酶活性的其他输入马达成分包括Tim44、Pam18和Pam16。Tim44协调输入马达在易位子通道上的定位，促进多肽底物在跨内膜易位时的结合。Pam18是一种特殊的J蛋白，可以刺激Ssc1的atp酶活性。有趣的是，Pam16与Pam18的J结构域序列相似，但不刺激Ssc1的atp酶活性。相反，Pam16与Pam18相互作用形成稳定的复合物，这是有效导入肽所必需的。这项研究旨在更好地了解这种对细胞存活至关重要的Pam18:Pam16异二聚体的作用。我们将确定Pam18:Pam16活性构象的结构和功能，并研究其对Ssc1活性的调控，以确定这种调控机制的生理意义。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化研究。Pam18:Pam16异源二聚体与核心转座子的结合将通过重构TIM复合物来研究，这将使由于该复合物的膜结合组分的不溶解性而使许多研究变得困难。这些技术的发展将开启一项广泛的事业，以更好地了解内膜环境下蛋白质易位的整体机制。线粒体的正常功能对许多细胞过程至关重要，包括细胞发育、钙信号传导、细胞凋亡和衰老。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对线粒体生物发生至关重要，对公共卫生具有重要意义。

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.