Cargo Sorting and Intralumenal Vesicle Budding by the ESCRT Complexes

通过 ESCRT 复合体进行货物分选和腔内囊泡出芽

• 批准号: 8349733
• 负责人: James Hurley
• 金额: $ 63.14万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349733
• 关键词: ATP phosphohydrolase; Binding; C-terminal; Cell division; Cell membrane; Cellular biology; Clathrin; Complex; Crystallization; Crystallography; Cytokinesis; Cytosol; Data; Dynamin; Electron Microscopy; Electron Transport Complex III; Electrons; Endocytosis; Endosomes; Energy Transfer; Eukaryota; Eukaryotic Cell; Event; Guanosine Triphosphate; HIV-1; Hydrolase; Hydrolysis; Intracellular Transport; Lysosomes; Mammals; Mechanics; Mediating; Membrane; Membrane Proteins; Microscopic; Mitochondria; Molecular; Molecular Conformation; Multivesicular Body; N-terminal; Neck; Pathway interactions; Phase; Plasma Cells; Population; Process; Proteins; Recruitment Activity; Secretory Vesicles; Shapes; Solutions; Sorting - Cell Movement; Spectrum Analysis; Spin Labels; Structure; Techniques; Transport Vesicles; Vesicle; Viral; Virus; Yeasts; endosome membrane; flexibility; monomer; phosphatidylinositol 3-phosphate; prevent; simulation; single molecule

Cargo Sorting and Intralumenal Vesicle Budding by the ESCRT Complexes Membrane budding and fission is a fundamental process of eukaryotic cell biology. Endocytosis, the formation of intracellular transport and secretory vesicles, and mitochondrial fission are examples of inward budding. In the classical example of clathrin-mediated endocytosis, the cytosolic protein dynamin forms arrays on the outside of the membrane neck, and membrane fission is driven thermodynamically by the hydrolysis of GTP. The formation of multivesicular bodies (MVBs) is the prototypical example of outward budding. MVBs are formed during the maturation of endosomes destined to fuse with lysosomes, and mediate the sorting of ubiquitinated membrane proteins to the lysosome. Portions of the limiting membrane of the endosome are internalized to form intralumenal vesicles (ILVs). When the MVB fuses with the lysosome, ILV contents are degraded by lysosomal hydrolases. When ILVs are released through fusion with the plasma membrane, they are referred to as exosomes. The budding of enveloped viruses from the plasma membrane and cell division (cytokinesis) are other examples of outward budding events. Outward budding events in MVB formation, viral budding, and cytokinesis are directed from the cytosol. Since cytosol is in contact with the inside, not the outside of the neck of the nascent bud, the mechanics of membrane fission differ fundamentally from inward budding, and utilize a completely distinct protein machinery. A major breakthrough in understanding outward budding came from the identification in yeast of the ESCRT machinery responsible for MVB formation. The ESCRT machinery is conserved throughout eukaryotes, and many enveloped viruses of mammals use the ESCRT pathway to bud, including HIV-1. The closure of the membrane neck in cytokinesis also uses the ESCRT pathway. The assembly of ESCRT complexes on endosomes is triggered by the presence of phosphatidylinositol 3-phosphate (PI(3)P) and ubiquitinated cargo proteins. ESCRT-I and II directly bind to cargo, and in turn recruit ESCRT-III. There are four ESCRT-III subunits in yeast, Vps2, Vps20, Vps24, and Snf7, together with two associated ESCRT-III-like proteins, Did2 and Vps60. ESCRT-III subunits exist in the cytosol as monomers, and assemble with each other on membranes in large multimeric arrays. ESCRT-II is a Y-shaped complex that contains two copies of the Vps25 subunit, which recruits ESCRT-III by directly binding to Vps20. Vps20 binds to Snf7, comprising a subcomplex of ESCRT-III. Snf7, in turn, directly binds to the Bro1 domain of the ESCRT-associated protein Alix (known as Bro1 in yeast). The Vps20:Snf7 complex recruits the Vps2:Vps24 subcomplex to form the complete ESCRT-III complex. A subset of ESCRT-III proteins directly bind to the N-terminal MIT domain of the AAA ATPase Vps4. Vps4 is a central player in the MVB pathway that is required for the disassembly of the ESCRT-III complex. ESCRT function can be conceptually separated into two phases: cargo recruitment and concentration, followed by membrane invagination and budding. The long term objectives of this project are to: 1) determine the structures of ESCRT complexes by x-ray crystallography, abetted where necessary by electron microscopy, hydrodynamics, molecular simulations, and small angle x-ray scattering; 2) to determine how ESCRTs assemble on membranes containing PI(3)P and cargo using binding and spectroscopic techniques; and 3) to study the mechanism of ILV formation by a microscopic, spectroscopic, and structure/function approaches. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37, and Mvb12. The crystal structures of the core complex and the UEV and Vps28 C-terminal (CTD) domains have been determined, but internal flexibility has prevented crystallization of intact ESCRT-I. Over the past FY, we have characterized the structure of ESCRT-I in solution by simultaneous structural refinement against small angle x-ray scattering (SAXS) and double electron-electron resonance (DEER) spectroscopy of spin labeled complexes. An ensemble of at least six structures, comprising an equally populated mixture of closed and open conformations, was necessary to fit all of the data. This structural ensemble was cross-validated against single molecule Frster resonance energy transfer (FRET) spectroscopy, which suggested the presence of a continuum of open states. ESCRT-I in solution thus appears to consist of a 50 % population of one or a few related closed conformations, with the other 50 % populating a continuum of open conformations. These conformations provide references points for the structural pathway by which ESCRT-I induces membrane buds.

通过 ESCRT 复合体进行货物分选和腔内囊泡出芽 膜出芽和分裂是真核细胞生物学的基本过程。内吞作用、细胞内运输和分泌囊泡的形成以及线粒体裂变是向内出芽的例子。在网格蛋白介导的内吞作用的经典例子中，胞浆蛋白动力在膜颈的外侧形成阵列，并且膜裂变由 GTP 的水解热力学驱动。多泡体（MVB）的形成是向外出芽的典型例子。 MVB 是在内体成熟过程中形成的，注定要与溶酶体融合，并介导泛素化膜蛋白向溶酶体的分选。内体的部分限制膜被内化形成腔内囊泡（ILV）。当 MVB 与溶酶体融合时，ILV 内容物被溶酶体水解酶降解。当 ILV 通过与质膜融合释放时，它们被称为外泌体。有包膜病毒从质膜出芽和细胞分裂（细胞分裂）是向外出芽事件的其他例子。 MVB 形成、病毒出芽和胞质分裂中的向外出芽事件是由细胞质指导的。由于胞质溶胶与新生芽的颈部内部而不是外部接触，因此膜裂变的机制与向内出芽根本不同，并利用完全不同的蛋白质机制。理解外芽的重大突破来自于酵母中负责 MVB 形成的 ESCRT 机制的鉴定。 ESRT 机制在真核生物中是保守的，许多哺乳动物的有包膜病毒都使用 ESRT 途径来萌芽，包括 HIV-1。胞质分裂中膜颈的闭合也使用 ESCRT 途径。 ESCRT 复合物在内体上的组装是由磷脂酰肌醇 3-磷酸 (PI(3)P) 和泛素化货物蛋白的存在触发的。 ESCRT-I 和 II 直接与货物结合，进而招募 ESCRT-III。酵母中有四个 ESCRT-III 亚基：Vps2、Vps20、Vps24 和 Snf7，以及两个相关的 ESCRT-III 样蛋白：Did2 和 Vps60。 ESCRT-III 亚基作为单体存在于细胞质中，并在膜上以大型多聚体阵列彼此组装。 ESCRT-II 是一种 Y 形复合物，包含两个 Vps25 亚基拷贝，它通过直接与 Vps20 结合来招募 ESCRT-III。 Vps20 与 Snf7 结合，构成 ESCRT-III 的子复合物。 Snf7 反过来又直接与 ESCRT 相关蛋白 Alix 的 Bro1 结构域（在酵母中称为 Bro1）结合。 Vps20:Snf7 复合体招募 Vps2:Vps24 子复合体形成完整的 ESCRT-III 复合体。 ESCRT-III 蛋白的一个子集直接结合 AAA ATPase Vps4 的 N 端 MIT 结构域。 Vps4 是分解 ESCRT-III 复合物所需的 MVB 途径的核心参与者。 ESCRT 功能在概念上可以分为两个阶段：货物招募和浓缩，然后是膜内陷和出芽。该项目的长期目标是：1）通过 X 射线晶体学确定 ESCRT 复合物的结构，必要时辅以电子显微镜、流体动力学、分子模拟和小角度 X 射线散射； 2) 使用结合和光谱技术确定 ESCRT 如何在含有 PI(3)P 和货物的膜上组装； 3）通过微观、光谱和结构/功能方法研究ILV形成机制。 ESCRT-I 是 Vps23、Vps28、Vps37 和 Mvb12 的异四聚体。核心复合物以及 UEV 和 Vps28 C 末端 (CTD) 结构域的晶体结构已确定，但内部柔性阻碍了完整 ESCRT-I 的结晶。在过去的一个财年中，我们通过对自旋标记复合物的小角 X 射线散射 (SAXS) 和双电子-电子共振 (DEER) 光谱同时进行结构精修，表征了溶液中 ESCRT-I 的结构。为了拟合所有数据，需要一个至少由六个结构组成的整体，其中包括同等数量的封闭和开放构象的混合物。这种结构整体与单分子弗斯特共振能量转移（FRET）光谱进行了交叉验证，这表明存在连续的开放态。因此，溶液中的 ESCRT-I 似乎由一个或几个相关闭合构象的 50% 群体组成，另外 50% 则由开放构象的连续体组成。这些构象为 ESCRT-I 诱导膜芽的结构途径提供了参考点。