Quality control of mislocalized membrane proteins

项目摘要 细胞膜结合区室如内质网的特性和功能 (ER)线粒体和突触囊泡，在很大程度上由蛋白质组成决定。细胞器 功能障碍和受损的膜蛋白质质量控制（QC）是神经变性疾病的标志， 包括肌萎缩侧索硬化症（ALS）、阿尔茨海默氏症、帕金森氏症和亨廷顿氏病。分子- 需要深入了解确保高保真膜蛋白生物合成的机制， 了解神经退行性疾病如何发展并确定有效的治疗方法。所有膜蛋白 面临两个基本的生物合成挑战。首先，它们必须定位于正确的细胞膜。 其次，它们必须以正确的方式将疏水跨膜结构域插入靶膜中。 导向目前尚不清楚细胞如何满足这些基本的生物合成要求的多样性膜 占蛋白质组的25-30%。使用单跨膜蛋白作为模型，我们有 建立了膜蛋白生物合成和QC的实验系统，这些系统易于进行机理分析， 解剖通过这些系统，我们最近发现ER驻留转运蛋白ATP 13 A1脱位， 错误定位的线粒体膜蛋白。通过ATP 13 A1的蛋白质移位提供了机会， 正确的靶向，并需要维持线粒体蛋白定位。在本提案中，我们将利用 ATP 13 A1作为一个分子把手，研究导致、识别和消除 异常蛋白质在目标1中，我们将确定异常插入的生物合成因子， 线粒体膜蛋白进入ER，并确定这些机制如何有助于 线粒体蛋白质稳态在目标2中，我们将研究选择性识别 并靶向错误定位的线粒体膜蛋白用于ER相关降解（ERAD）。在目标3中， 将研究II型膜蛋白的拓扑发生，这些蛋白应该插入ER， 在胞质溶胶中的末端。由于II型蛋白的一个亚类被ATP 13 A1消耗选择性地去稳定， 我们假设这些蛋白质具有易于以错误方向插入的特定特征， 需要ATP 13 A1介导的脱位。该项目的完成将揭示机制，错误的- 将膜蛋白插入ER，产生哺乳动物ERAD途径的机制模型，和 阐明了少数生物合成和质量控制因素如何处理大量和多样化的客户。总之， 这些发现将揭示膜蛋白QC靶点选择和机制的分子水平的见解 细胞生物合成和质量控制机制如何合作，以确保 膜蛋白的生物合成需要保持神经元的功能。

PROJECT SUMMARY The identities and functions of cellular membrane-bound compartments such as the endoplasmic reticulum (ER), mitochondria, and synaptic vesicles, are largely determined by protein composition. Organelle dysfunction and impaired membrane protein quality control (QC) are hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's, Parkinson's, and Huntington's disease. Molecular- level insights into the mechanisms that ensure high-fidelity membrane protein biogenesis are required to understand how neurodegenerative diseases develop and identify effective treatments. All membrane proteins face two fundamental biosynthetic challenges. First, they must localize to the correct cellular membrane. Second, they must insert hydrophobic transmembrane domains into target membranes in the correct orientation. It is not known how cells meet these basic biosynthetic requirements for the diverse membrane proteins that make up 25-30% of the proteome. Using single-spanning membrane proteins as models, we have established experimental systems of membrane protein biosynthesis and QC that are tractable for mechanistic dissection. With these systems, we recently discovered that the ER-resident transporter ATP13A1 dislocates mislocalized mitochondrial membrane proteins. Protein dislocation by ATP13A1 provides opportunities for correct targeting and is required to maintain mitochondrial protein localization. In this proposal, we will leverage ATP13A1 function as a molecular handle to study the mechanisms that lead to, recognize, and eliminate aberrant proteins at the ER. In Aim 1, we will identify the biosynthetic factors that aberrantly insert mitochondrial membrane proteins into the ER and determine how these mechanisms contribute to mitochondrial protein homeostasis. In Aim 2, we will investigate the QC mechanisms that selectively recognize and target mislocalized mitochondrial membrane proteins for ER-associated degradation (ERAD). In Aim 3, we will investigate the topogenesis of type II membrane proteins that should insert into the ER with their N- terminus in the cytosol. Because a subset of type II proteins is selectively destabilized by ATP13A1 depletion, we hypothesize that these proteins harbor specific features prone to insertion in the wrong orientation, resulting in the need for ATP13A1-mediated dislocation. Completion of this project will reveal mechanisms that mis- insert membrane proteins into the ER, generate a mechanistic model of a mammalian ERAD pathway, and shed light on how a handful of biosynthetic and QC factors handle a large and diverse clientele. Altogether, these findings will reveal molecular-level insights into membrane protein QC target selection and mechanistic principles underlying how cellular biosynthetic and QC mechanisms collaborate to ensure the integrity of membrane protein biogenesis needed to preserve neuronal function.