Molecular Basis of Tail-Anchored Membrane Protein Targeting

描述（由申请人提供）：该项目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，该途径指导尾锚定（TA）膜蛋白插入内质网（ER）膜。TA蛋白构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，从细胞内运输和病毒复制到细胞死亡的调节。尽管这些蛋白在生理上具有重要意义，但介导TA蛋白识别、靶向和插入正确细胞器膜的机制和分子机制尚不清楚。新合成的蛋白靶向和插入内质网膜是一个必要的细胞过程。对于大多数膜蛋白来说，这是通过信号识别粒子介导的共翻译过程实现的。然而，对于近5%的尾锚定真核膜蛋白，靶向是在翻译后实现的。这一过程是由一种新发现的进化上保守的atp酶介导的，称为TRC40，它与er结合受体相互作用，以确保有效和准确的TA蛋白靶向。在一个重要的突破中，我们最近确定了TRC40的高分辨率晶体结构。基于这些结构信息，我们将检验以下具体假设。1) TRC40对靶向信号的特异性理化性质的识别指导了TA蛋白的选择性靶向。2) TRC40的构象变化调节其atp酶活性和TA底物相互作用。3) TRC受体通过与TRC40-TA底物复合物的特异性相互作用催化这些构象变化。利用强大的跨学科方法，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。探讨trc402选择性识别TA底物的分子基础。确定TRC40构象变化如何调节atp酶活性和TA底物相互作用3。建立TRC受体如何协调内质网膜上TA蛋白的靶向和释放

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 PUBLIC HEALTH RELEVANCE: Tail-anchored (TA) membrane proteins play critical roles in virtually all aspects of cell biology. Given the role of TA proteins in pathologies ranging from cancer, neurodegenerative disease and chronic liver diseases (to name only a few), it is clear that a detailed mechanistic understanding of the targeting, membrane insertion and regulation of TA proteins is of fundamental cell biological and physiological significance. The studies described here will provide insight into the molecular details of TA membrane protein biogenesis, and may ultimately lead to the development of new therapeutic strategies that work by modulating the targeting of TA membrane proteins.