Molecular Basis of Tail-Anchored Membrane Protein Targeting

描述（由申请人提供）：本研究的目标是对新发现的指导尾锚定（TA）膜蛋白插入内质网（ER）膜的翻译后靶向途径进行详细的分子理解。TA蛋白占所有真核生物膜蛋白的近5%，通过单个C-末端跨膜结构域（TMD）锚定到脂质双层，并且具有面向胞质的氨基末端结构域。这些蛋白质存在于几乎所有的细胞膜中，它们介导许多重要的细胞过程。 TA蛋白的翻译后靶向和插入由新发现的“尾锚定蛋白的引导进入”（GET）途径介导。在过去几年中，已经确定了这一途径的基本框架。首先，“预靶向”因子捕获胞质溶胶中新合成的TA蛋白，并将其转移到称为Get 3的可溶性ATP酶。这种靶向复合物通过与Get 1/2受体复合物的相互作用被导向ER膜，这对于驱动TA底物释放和插入膜是必要的和足够的。 我们的研究目标是确定TA蛋白插入的生物化学和生物物理学原理。首先，我们将阐明Get 3-TA底物靶向复合物在靶向途径的限定步骤沿着的分子身份（目的1）。其次，我们将确定TA底物是否直接整合到双层中，或者插入是否需要Get 1/2的陪伴作用。 受体复合物（Aim 2）。最后，我们将定义Get 1/2复合物的静态和动态特性如何使其能够协调TA底物招募，释放和插入的基本步骤（目的3）。这些目标将使用强大的跨学科方法来实现，该方法将结构功能分析与最先进的光谱研究相结合。 通过定义已知插入途径之间的共同主题，这项工作将加深我们对TMD插入的基本细胞生物学和生物物理过程的理解。通过开发新的工具和实验策略，这项工作有望使其他复杂的膜相关过程的分析。最后，由于TA蛋白生物发生的缺陷与许多人类病理学有关，这些研究有望带来新的治疗策略，用于对抗人类疾病。

DESCRIPTION (provided by applicant): The goal of this research is to develop a detailed molecular understanding of a newly discovered post-translational targeting pathway that directs insertion of tail-anchored (TA) membrane proteins into the endoplasmic reticulum (ER) membrane. TA proteins, which account for nearly 5% of all eukaryotic membrane proteins, are anchored to the lipid bilayer by a single C-terminal transmembrane domain (TMD) and have a cytosolic-facing amino-terminal domain. These proteins are found in virtually all cell membranes where they mediate numerous essential cellular processes. Post-translational targeting and insertion of TA proteins is mediated by the newly discovered 'guided entry of tail-anchored protein' (GET) pathway. Over the past few years, a basic framework for the pathway has been defined. First, a 'pre-targeting' factor captures a newly synthesized TA protein in the cytosol, and transfers it to a soluble ATPase, called Get3. This targeting complex is directed to the ER membrane via an interaction with the Get1/2 receptor complex which is both necessary and sufficient to drive TA substrate release and insertion into the membrane. Our research goal is to define the biochemical and biophysical principles that underlie TA protein insertion. First, we will elucidate the molecular identity of the Get3-TA substrate targeting complex at defined steps along the targeting pathway (Aim 1). Second, we will determine whether TA substrates are integrated directly into the bilayer or if insertion requires a chaperoning role for the the Get1/2 receptor complex (Aim 2). Finally, we will define how the static and dynamic properties of the Get1/2 complex allow it to orchestrate the essential steps of TA substrate recruitment, release and insertion (Aim 3). These Aims will be accomplished using a powerful interdisciplinary approach that combines structure-function analyses with state-of- the-art spectroscopic studies. By defining common themes between known insertion pathways, this work will deepen our understanding of the fundamental cell biological and biophysical process of TMD insertion. By developing new tools and experimental strategies, this work promises to enable analysis of other complex membrane-associated processes. Finally, because defects in TA protein biogenesis are linked to much human pathology, these studies promise insight that may lead to new therapeutic strategies for use in the fight against human disease.