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翻译后靶向途径进行详细的分子理解，该途径指导尾锚定（TA）膜蛋白插入内质网（ER）膜。TA蛋白占所有真核生物膜蛋白的近5%，通过单个c端跨膜结构域（TMD）锚定在脂质双分子层上，并具有面向细胞质的氨基末端结构域。这些蛋白存在于几乎所有的细胞膜中，介导许多基本的细胞过程。TA蛋白的翻译后靶向和插入是由新发现的“尾锚蛋白引导进入”（GET）途径介导的。在过去几年中，这一途径的基本框架已经确定。首先，一个“预靶向”因子捕获细胞质中新合成的TA蛋白，并将其转移到一种名为Get3的可溶性atp酶。这种靶向复合物通过与Get1/2受体复合物的相互作用被引导到内质网膜，这是驱动TA底物释放和插入膜的必要和充分条件。我们的研究目标是确定TA蛋白插入背后的生化和生物物理原理。首先，我们将沿着靶向途径的特定步骤阐明Get3-TA底物靶向复合物的分子特性（目的1）。其次，我们将确定TA底物是否直接集成到双分子层中，或者插入是否需要Get1/2的陪伴作用

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.