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分裂是向内出芽的例子。在网格蛋白介导的内吞作用的经典例子中，胞质蛋白动力蛋白在膜颈的外侧形成阵列，并且膜分裂由GTP的水解驱动。多泡体（MVB）的形成是向外出芽的典型例子。MVB在注定与溶酶体融合的内体成熟期间形成，并介导泛素化膜蛋白向溶酶体的分选。内体的部分界膜被内化以形成腔内囊泡（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通过与质膜融合而释放时，它们被称为外来体。包膜病毒从质膜上出芽和细胞分裂（胞质分裂）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外显出芽事件由胞质溶胶引导。由于胞质溶胶是与内部接触，而不是与新生芽的颈部外部接触，因此膜分裂的机制与向内出芽的机制根本不同，并且利用完全不同的蛋白质机制。在理解向外出芽方面的一个重大突破来自于在酵母中鉴定负责MVB形成的ESCRT机制。ESCRT机制在整个真核生物中是保守的，许多哺乳动物的包膜病毒使用ESCRT途径出芽，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，反过来招募ESCRT-III。在酵母中有四个ESCRT-III亚基，Vps 2，Vps 20，Vps 24和Snf 7，以及两个相关的ESCRT-III样蛋白，Did 2和Vps 60。ESCRT-III亚基作为单体存在于胞质溶胶中，并在大的多聚体阵列中在膜上彼此组装。ESCRT-II是一种Y形复合物，含有两个拷贝的Vps 25亚基，通过直接结合Vps 20招募ESCRT-III。Vps 20与Snf 7结合，包含ESCRT-III的亚复合物。Snf 7又直接与ESCRT相关蛋白阿利克斯（在酵母中称为Bro 1）的Bro 1结构域结合。Vps 20：Snf 7复合物募集Vps 2：Vps 24亚复合物以形成完整的ESCRT-III复合物。ESCRT-III蛋白的一个子集直接结合到AAA ATP酶Vps 4的N-末端MIT结构域。Vps 4是MVB途径中的核心参与者，其是ESCRT-III复合物分解所需的。ESCRT功能可以在概念上分为两个阶段：货物招募和浓度，然后是膜内陷和出芽。本项目的长期目标是：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、流体力学、分子模拟和小角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）从微观、光谱、结构/功能等方面研究ILV的形成机制。 ESCRT-I是Vps 23、Vps 28、Vps 37和Mvb 12的异源四聚体。的核心复合物和UEV和Vps 28 C-末端（CTD）域的晶体结构已被确定，但内部的灵活性已阻止完整的ESCRT-I的结晶。在过去的FY，我们的特点是ESCRT-I在溶液中的结构，同时对小角X射线散射（SAXS）和双电子-电子共振（DEER）光谱的自旋标记复合物的结构细化。一个至少有六个结构的集合，包括一个相等的填充混合物的封闭和开放的构象，是必要的，以适应所有的数据。这种结构合奏交叉验证对单分子Frster共振能量转移（FRET）光谱，这表明存在一个连续的开放状态。因此，溶液中的ESCRT-I似乎由50%的一种或几种相关的闭合构象组成，而另外50%的群体是开放构象的连续体。这些构象为ESCRT-I诱导膜芽的结构途径提供了参考点。