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.

ESCRT复合物的货物排序和肉体内囊泡发芽 膜出芽和裂变是真核细胞生物学的基本过程。内吞作用，细胞内转运和分泌囊泡的形成以及线粒体裂变是内向萌芽的例子。在网格蛋白介导的内吞作用的经典例子中，胞质蛋白动力蛋白动力蛋白在膜颈外面形成阵列，膜裂变是由GTP的水解在热力学上驱动的。多囊体（MVB）的形成是外向萌芽的典型例子。 MVB是在注定与溶酶体融合的内体成熟过程中形成的，并介导泛素化膜蛋白的分选为溶酶体。内体的限制膜的一部分被内在形成赤囊（ILV）。当MVB与溶酶体融合时，ILV含量会被溶酶体水解酶降解。当通过与质膜融合释放ILV时，它们被称为外泌体。质膜和细胞分裂（细胞因子）的包膜病毒的萌芽是外向发芽事件的其他例子。 MVB形成，病毒萌芽和细胞因子的外向发芽事件是从细胞质引导的。由于细胞质与内部接触，而不是新生芽的颈部外部，因此膜裂变的力学与内向萌芽根本不同，并使用了完全不同的蛋白质机制。理解外向的一个重大突破来自负责MVB形成的ESCRT机制的鉴定。 ESCRT机械在整个真核生物中都是保守的，许多哺乳动物的包膜病毒都使用芽途径，包括HIV-1。细胞因子中膜颈的闭合也使用了ESCRT途径。 磷脂酰肌醇3-磷酸（PI（3）P）和泛素化的货物蛋白的存在触发了内体上ESCRT复合物的组装。 ESCRT-I和II直接与货物结合，进而募集ESCRT-III。酵母，VPS2，VPS20，VPS24和SNF7中有四个ESCRT-III亚基，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，并在大型多聚体阵列中相互组装。 ESCRT-II是一种Y形复合物，包含两个VPS25亚基的副本，该副本通过直接与VPS20结合来募集ESCRT-III。 VPS20与SNF7结合，包括ESCRT-III的子复合。 SNF7反过来直接与ESCRT相关蛋白ALIX的BRO1结构（称为酵母中的Bro1）结合。 VPS20：SNF7复合物募集VPS2：VPS24子复合物形成完整的ESCRT-III复合物。 ESCRT-III蛋白的子集直接与AAA ATPase VPS4的N端MIT结构域结合。 VPS4是MVB途径中的中心参与者，这是ESCRT-III综合体拆卸所需的。概念上可以将ESCRT功能分为两个阶段：货物募集和浓度，然后进行膜的内陷和萌芽。该项目的长期目标是：1）通过X射线晶体学确定ESCRT络合物的结构，在必要时通过电子显微镜，水动力学，分子模拟和小角度X射线散射来教to。 2）确定如何使用结合和光谱技术在包含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形成的机理。 ESCRT-I是VPS23，VPS28，VPS37和MVB12的异光学器。已经确定了核心复合物以及UEV和VPS28 C末端（CTD）结构域的晶体结构，但内部柔韧性阻止了完整的ESCRT-I结晶。在过去的FY中，我们通过针对小角度X射线散射（SAX）和双电子电子共振（DEER）光谱的旋转复合物的同时进行结构改进来表征溶液中ESCRT-I的结构。至少六个结构组成的合奏包括封闭和开放构象的同样构成的混合物，以适应所有数据。该结构合奏通过单分子Frster共振能量转移（FRET）光谱进行了交叉验证，这表明存在开放状态的连续体。因此，溶液中的ESCRT-I似乎由一个或几个相关的封闭构象的50％人口组成，而其他50％的人口则占据了开放构象的连续体。这些构象为ESCRT-I诱导膜芽的结构途径提供了参考点。