Mechanisms of membrane homeostasis through protein and lipid transport

项目摘要/摘要 真核细胞必须保持质膜的特定蛋白质和脂肪成分，以及所有 内膜结合细胞器，以便正常运作。即使膜生物发生是 对生命至关重要的是，建立膜的组成和组织的机制仍然很差 明白了。我们研究了膜不对称是如何形成的，这是真核细胞的一个基本特征。 质膜由磷脂酰丝氨酸和磷脂酰乙醇胺在细胞内的浓缩所定义 胞质小叶，而鞘磷脂和卵磷脂通常富含在细胞外小叶中。 双层的。调节细胞外叶上PS和PE的暴露有助于细胞信号转导， 胞质分裂、血液凝固、细胞-细胞融合、凋亡细胞身体清除和宿主与病毒的相互作用。膜 不对称性是由IV型P型ATPase(P4-ATPase)驱动的，P4-ATPase是一大家族的翻转酶 从膜双层的胞外小叶到胞内小叶的脂类。P4-ATPase亚家族是 在真核生物中高度保守，这些转运蛋白与病理条件有关 如肥胖相关2型糖尿病、心血管疾病、肝病、听力损失、免疫缺陷 以及严重的神经系统疾病。此外，P4-ATPase是囊泡介导的 高尔基复合体和内体系统内的蛋白质运输机制。通过它们在蛋白质中的作用 运输，P4-ATPase帮助控制细胞膜高尔基复合体的精确蛋白质组成 内质体和溶酶体。拟议的研究将确定P4-ATPase如何识别和 利用结构、生化和分子技术转运脂质底物以建立膜的不对称性 遗传方法。这些结构/功能研究将包括P4-ATPase活性是如何由POST调节的。 翻译修饰和蛋白质-蛋白质相互作用。我们还将探讨无线网络的需求 特定底物脂类的运输，如葡萄糖神经酰胺和磷脂酰丝氨酸，对细胞形态发生的影响 真菌致病机制、营养信号和蛋白质运输。对于后一项研究，我们将探讨P4是如何- ATPase有助于驱动囊泡介导的蛋白质运输，重点是由COPI和逆转录聚体形成的载体。 泛素化在依赖P4-ATPase和COPI的转运途径中的非典型作用也将是 已定义。总而言之，这些研究应该会更好地理解P4-ATPase是如何发挥其作用的 基本功能，对我们理解和最终治疗相关病理的能力将是无价的 伴有P4-ATPase缺乏症。

Project Summary/Abstract Eukaryotic cells must maintain a specific protein and lipid composition of the plasma membrane and all of the internal membrane-bound organelles in order to function normally. Even though membrane biogenesis is crucial for life, mechanisms for establishing the composition and organization of membranes remain poorly understood. We study how membrane asymmetry is established, a fundamental feature of the eukaryotic cell plasma membrane defined by the enrichment of phosphatidylserine and phosphatidylethanolamine within the cytosolic leaflet, while sphingolipids and phosphatidylcholine are typically enriched in the extracellular leaflet of the bilayer. Regulated exposure of PS and PE on the extracellular leaflet contributes to cell signaling, cytokinesis, blood clotting, cell-cell fusion, apoptotic cell corpse removal and host-viral interactions. Membrane asymmetry is driven by type IV P-type ATPases (P4-ATPases), which are a large family of flippases that pump lipids from the extracellular leaflet to the cytosolic leaflet of the membrane bilayer. The P4-ATPase subfamily is highly conserved among eukaryotes and these transporters have been implicated in pathological conditions such as obesity-linked type 2 diabetes, cardiovascular disease, liver disease, hearing loss, immune deficiency, and severe neurological disease. In addition, P4-ATPases are critical components of the vesicle-mediated protein trafficking machinery within the Golgi complex and endosomal system. Through their role in protein trafficking, P4-ATPases help control the precise protein composition of the plasma membrane, Golgi complex endosomes and lysosomes. The proposed studies will determine how the P4-ATPases recognize and transport their lipid substrates to establish membrane asymmetry using structural, biochemical and molecular genetic approaches. These structure/function studies will include how P4-ATPase activity is regulated by post- translational modification and protein-protein interactions. We will also probe the cellular requirements for transport of specific substrate lipids, like glucosylceramide and phosphatidylserine, on cell morphogenesis, fungal pathogenesis, nutrient signaling, and protein trafficking. For the latter studies, we will probe how P4- ATPases help drive vesicle-mediated protein transport with a focus on carriers formed by COPI and retromer. Atypical roles for ubiquitination in the P4-ATPase- and COPI-dependent transport pathways will also be defined. In total, these studies should lead to a much better understanding of how P4-ATPases exert their essential function, and will be invaluable to our ability to understand and ultimately treat pathologies associated with P4-ATPase deficiency.