Molecular mechanisms of post-translational targeting of tail-anchored proteins.

描述(申请人提供)：膜蛋白约占细胞蛋白质组的30%，其有效和准确的定位对所有细胞的结构和正常功能至关重要。与研究较好的共翻译蛋白靶向途径相比，翻译后膜蛋白靶向是额外的挑战，因为底物蛋白上存在高度疏水的跨膜结构域。破译分子策略将这些易于聚集的底物护送到正确的目标位置是一个根本的机械挑战。在引导进入尾锚(GET)途径中，一系列复杂的蛋白质相互作用介导TA蛋白翻译后递送到内质网膜，为解决这些问题提供了极好的机会。我们的总体目标是在生化和生物物理水平上破译这种新途径靶向TA蛋白的分子机制。我们的具体目标是了解该途径中的中心ATPase Get3如何利用其ATPase循环来驱动和协调GET途径中复杂的蛋白质相互作用级联反应。为此，我们将为Get3ATPase周期建立一个准确的框架，并确定在这个周期中发生的构象变化。我们将定义Get3的上下游相互作用伙伴何时、何地以及如何调节其ATPase循环，并反过来，该ATPase循环如何驱动Get3与其效应蛋白的有序级联相互作用。我们将开发新的分析方法来实时剖析靶向反应的各个步骤，并利用这一方法来破译该途径如何实现高度特异性的底物选择。这些研究将极大地促进我们对膜蛋白翻译后靶向的分子机制的理解。此外，Get3代表了第一个真正的ATPase，属于一类新的‘二聚化激活的’核苷酸水解酶；对这种ATPase二聚体的研究将有助于测试、扩展和推广这一日益增长的新型细胞调节因子的调节原理。

DESCRIPTION (provided by applicant): Membrane proteins comprise ~30% of a cell's proteome, and their efficient and accurate localization is essential for the structure and proper functioning of all cells. Compared to the well-studied co-translational protein targeting pathway, post-translational membrane protein targeting poses additional challenges due to the presence of highly hydrophobic transmembrane domains on the substrate protein. Deciphering the molecular strategies to escort such aggregation-prone substrates to the correct target site is a fundamental mechanistic challenge. In the Guided Entry of Tail-anchor (GET) pathway, a complex cascade of protein interactions mediates the post-translational delivery of TA proteins to the endoplasmic reticulum membrane, providing an excellent opportunity to address these questions. Our general goal is to decipher, at the biochemical and biophysical level, the molecular mechanisms underlying the targeting of TA proteins by this novel pathway. Our specific goal is to understand how Get3, the central ATPase in this pathway, uses its ATPase cycle to drive and coordinate the complex cascade of protein interactions during the GET pathway. To this end, we will establish a precise framework for the Get3 ATPase cycle and identify conformational changes that occur during this cycle. We will define when, where and how the upstream and downstream interaction partners of Get3 regulate its ATPase cycle and reciprocally, how this ATPase cycle drives an ordered cascade of interactions of Get3 with its effector proteins. We will develop novel assays to dissect individual steps of the targeting reaction in real time and use this to decipher how highly specific substrate selection is achieved by the pathway. These studies will significantly advance our understanding of the molecular mechanisms that underlie the post- translational targeting of membrane proteins. Further, Get3 represents the first eukaryotic ATPase that belongs to a novel class of 'dimerization-activated' nucleotide hydrolases; studies of this ATPase dimer will be instrumental to test, expand, and generalize the regulatory principles for this growing class of novel cellular regulators.