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

描述（由申请人提供）：膜蛋白占细胞蛋白质组的约30%，其有效和准确的定位对于所有细胞的结构和正常功能至关重要。与充分研究的共翻译蛋白靶向途径相比，翻译后膜蛋白靶向由于底物蛋白上存在高度疏水的跨膜结构域而带来了额外的挑战。破译护送这种聚集倾向的基板到正确的靶位点的分子策略是一个基本的机械挑战。在引导进入尾锚（GET）途径中，蛋白质相互作用的复杂级联介导TA蛋白质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。我们的总体目标是破译，在生物化学和生物物理水平上，TA蛋白的靶向通过这种新的途径的分子机制。我们的具体目标是了解Get 3，在这个途径中的中央ATP酶，如何使用其ATP酶循环来驱动和协调GET途径中蛋白质相互作用的复杂级联。为此，我们将建立一个精确的框架Get 3 ATP酶周期，并确定在此周期中发生的构象变化。我们将定义何时，何地以及如何上游和下游的相互作用伙伴的Get 3调节其ATP酶循环和ATP酶，这个ATP酶循环如何驱动一个有序的级联反应的Get 3与其效应蛋白的相互作用。我们将开发新的检测方法，在真实的时间内剖析靶向反应的各个步骤，并利用这些方法来破译该途径如何实现高度特异性的底物选择。这些研究将大大推进我们对膜蛋白翻译后靶向的分子机制的理解。此外，Get 3代表了第一个真核细胞ATP酶，属于一类新的“二聚化激活”的核苷酸水解酶;这种ATP酶二聚体的研究将有助于测试，扩大和推广这一类新的细胞调节剂的监管原则。

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.