Quality control of mislocalized membrane proteins

项目总结 内质网等细胞膜结合室的特性和功能 线粒体和突触小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量控制(QC)是神经退行性疾病的特征， 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症。分子- 需要对确保高保真膜蛋白生物发生的机制有更深入的了解 了解神经退行性疾病是如何发展的，并确定有效的治疗方法。所有膜蛋白 面临两个根本性的生物合成挑战。首先，它们必须定位在正确的细胞膜上。 其次，它们必须将疏水的跨膜结构域插入正确的靶膜中 定位。目前尚不清楚细胞如何满足不同细胞膜的基本生物合成要求。 构成蛋白质组25%-30%的蛋白质。使用单跨膜蛋白作为模型，我们有 建立了易于机械化的膜蛋白生物合成和质控实验体系 解剖。对于这些系统，我们最近发现内质网驻留转运体ATP13A1错位 线粒体膜蛋白定位错误。ATP13A1引起的蛋白质错位为 正确的靶向，是维持线粒体蛋白定位所必需的。在这项提案中，我们将利用 ATP13A1作为分子手柄研究导致、识别和消除的机制 内质网的异常蛋白质。在目标1中，我们将确定异常插入 线粒体膜蛋白进入内质网，并确定这些机制如何参与 线粒体蛋白质动态平衡。在目标2中，我们将研究选择性识别的QC机制 以及靶向错位线粒体膜蛋白以进行内质网相关降解(ERAD)。在目标3中，我们 将研究应该插入内质网的II型膜蛋白的拓扑发生。 胞质中的末端。因为ATP13A1耗尽选择性地破坏了类型II蛋白的子集的稳定性， 我们假设这些蛋白质含有特定的特征，容易插入错误的方向，导致 需要ATP13A1介导的错位。该项目的完成将揭示错误的机制-- 将膜蛋白插入内质网，产生哺乳动物ERAD途径的机制模型，以及 阐明了几个生物合成和质量控制因素是如何应对庞大而多样化的客户的。总而言之， 这些发现将在分子水平上揭示膜蛋白QC靶点的选择和机制 细胞生物合成和质量控制机制如何协作以确保 膜蛋白的生物发生需要保护神经元的功能。

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.