Molecular Basis of Tail-Anchored Membrane Protein Targeting

描述(由申请人提供)：该计划的总体目标是对一种新的翻译后靶向途径进行详细的分子理解，该途径引导尾部锚定(TA)膜蛋白插入内质网(ER)膜。TA蛋白组成了一个完整的膜蛋白大家族，在细胞生物学的几乎所有方面都发挥着关键作用，从细胞内转运和病毒复制到细胞死亡的调节。尽管这些蛋白具有重要的生理意义，但对介导TA蛋白识别、靶向和插入正确细胞膜的机制和分子机制知之甚少。靶向并将新合成的蛋白质插入内质网是一个重要的细胞过程。对于大多数膜蛋白来说，这是在信号识别颗粒介导的过程中通过共翻译实现的。然而，对于所有尾部锚定的真核膜蛋白中的近5%，靶向是在翻译后实现的。这个过程是由一个新发现的进化保守的ATPase介导的，称为TRC40，它与内质网结合的受体相互作用，确保有效和准确的TA蛋白靶向。在一项重大突破中，我们最近确定了TRC40的高分辨晶体结构。基于这一结构性信息，将检验以下具体假设。1)识别靶向信号的特定物理化学性质，指导TRC40选择性靶向TA蛋白。2)TRC40的构象变化调节其ATPase活性和TA底物的相互作用。3)TRC受体通过与TRC40-TA底物复合体的特异性相互作用来催化这些构象变化。利用一种强大的跨学科方法，我们将建立TA蛋白靶向和插入过程的基本生化和生物物理原理。本项目的具体目标是：1.确定TRC40选择性识别TA底物的分子基础2.确定TRC40的构象变化如何调节ATPase活性和TA底物相互作用3.建立TRC受体如何协调TA蛋白在内质网膜上的靶向和释放

DESCRIPTION (provided by applicant): The overall goal of this program is to develop a detailed molecular understanding of a novel post- translational targeting pathway that directs insertion of tail-anchored (TA) membrane proteins into the endoplasmic reticulum (ER) membrane. TA proteins constitute a large family of integral membrane proteins that play critical roles in virtually all aspects of cell biology, ranging from intracellular trafficking and viral replication to regulation of cell death. Despite the physiological importance of these proteins, the machinery and molecular mechanisms that mediate recognition, targeting, and insertion of TA proteins into the correct organellar membrane are poorly understood. The targeting and insertion of newly synthesized proteins into the ER membrane is an essential cellular process. For most membrane proteins, this is achieved co-translationally in a process mediated by the signal recognition particle. However, for the nearly 5% of all eukaryotic membrane proteins that are tail-anchored, targeting is achieved post-translationally. This process is mediated by a newly discovered and evolutionarily conserved ATPase, termed TRC40, which interacts with an ER-bound receptor to ensure efficient and accurate TA protein targeting. In an important breakthrough, we have recently determined high-resolution crystal structures of TRC40. Based upon this structural information, the following specific hypotheses will be tested. 1) Recognition of specific physiochemical properties of the targeting signal directs selective TA protein targeting by TRC40. 2) Conformational changes in TRC40 regulate its ATPase activity and TA substrate interactions. 3) The TRC receptor catalyzes these conformational changes through specific interactions with the TRC40-TA substrate complex. Using a powerful interdisciplinary approach, we will establish the fundamental biochemical and biophysical principles that underlie the process of TA protein targeting and insertion. The specific aims of this project are: 1. To determine the molecular basis of selective TA substrate recognition by TRC40 2. To determine how conformational changes in TRC40 regulate ATPase activity and TA substrate interactions 3. To establish how the TRC receptor coordinates targeting and release of TA proteins at the ER membrane