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: 8148740
• 负责人: James Hurley
• 金额: $ 36.33万
• 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/8148740
• 关键词: ATP phosphohydrolase; Binding; Complex; Crystallography; Cytokinesis; Cytosol; Electron Microscopy; Electron Transport Complex III; Endocytosis; Eukaryotic Cell; Guanosine Triphosphate; HIV-1; Hydrolase; Hydrolysis; In Vitro; Lysosomes; Mammals; Membrane Proteins; Microscopic; Mitochondria; Molecular; Multivesicular Body; Neck; Pathway interactions; Phase; Process; Shapes; Sorting - Cell Movement; Vesicle; cofactor; crosslink; endosome membrane; in vivo

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. In this reporting period, the biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, I, II, and III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction. The final step in the ESCRT cycle is the disassembly of the ESCRT-III lattice by the AAA ATPase Vps4. Vps4 assembles on its membrane-bound ESCRT-IIII substrate with its cofactor, Vta1. The crystal structure of the dimeric VSL domain of yeast Vta1 with the small ATPase and the b domains of Vps4 was determined. Residues involved in structural interactions are conserved and are required for binding in vitro and for Cps1 sorting in vivo. Modeling of the Vta1 complex in complex with the lower hexameric ring of Vps4 indicates that the 2-fold axis of the Vta1 VSL domain is parallel to within 20 degrees of the 6-fold axis of the hexamer. This suggests that Vta1 might not crosslink the two hexameric rings of Vps4, but rather stabilizes an array of Vps4-Vta1 complexes for ESCRT-III disassembly.

ESCRT复合体的货物分选和腔内囊泡萌发 膜的萌发和分裂是真核细胞生物学的基本过程。内吞作用、细胞内运输和分泌小泡的形成以及线粒体的分裂都是向内萌发的例子。在经典的网状蛋白介导的内吞作用中，胞内的蛋白动力蛋白在膜颈部的外侧形成阵列，膜的分裂是由GTP的水解热力学驱动的。多囊泡体(MVB)的形成是外萌发的典型例子。MVB是在内体成熟的过程中形成的，内体注定要与溶酶体融合，并介导泛素化的膜蛋白分选到溶酶体。内体限制膜的一部分内化形成腔内小泡(ILV)。当MVB与溶酶体融合时，ILV的含量被溶酶体水解酶降解。当ILV通过与质膜融合而释放时，它们被称为外体。被包裹的病毒从质膜上萌发和细胞分裂(胞质分裂)是向外萌发事件的其他例子。在MVB形成、病毒萌发和胞质分裂中的外向萌发事件是从细胞质中引导的。由于细胞质接触的是新芽的内部，而不是颈部的外部，因此膜分裂的机制从根本上不同于向内发芽，并利用了完全不同的蛋白质机制。在理解向外萌发方面的一个重大突破来自于在酵母中发现了负责MVB形成的ESCRT机制。ESCRT机制在真核生物中是保守的，许多哺乳动物的包膜病毒使用ESCRT途径萌发，包括HIV-1。胞质分裂中膜颈的关闭也使用ESCRT途径。 ESCRT复合体在内体上的组装是由磷脂酰肌醇3-磷酸(PI(3)P)和泛素化的Cargo蛋白触发的。ESCRT-I和ESCRT-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类似蛋白Did2和Vps60。ESCRT-III亚基以单体形式存在于胞浆中，并在大的多聚体阵列中相互组装在膜上。ESCRT-II是一个Y形复合体，包含Vps25亚单位的两个拷贝，它通过直接与Vps20结合来招募ESCRT-III。Vps20与Snf7结合，构成ESCRT-III的一个亚复合体。SNF7则直接与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亚复合体形成完整的ESCRT-III复合体。ESCRT-III蛋白的一部分直接与AAA ATPase Vps4的N端MIT结构域结合。Vps4是MVB途径中的中心角色，MVB途径是ESCRT-III复合体分解所必需的。ESCRT的功能可以概念性地分为两个阶段：货物募集和集中，随后是膜内陷和萌发。该项目的长期目标是：1)通过X射线结晶学确定ESCRT络合物的结构，必要时借助电子显微镜、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如何组装在含有PI(3)P和Cargo的膜上；以及3)通过显微、光谱和结构/功能方法研究ILV的形成机制。 在本报告所述期间，以巨大的单层囊泡、荧光ESCRT-0、I、II和III复合体以及膜系留的荧光泛素融合为模型货物，重组和可视化了多囊泡体的生物发生。ESCRT-0形成簇状货物的区域，但不使膜变形。ESCRT-I和ESCRT-II结合在一起使膜变形成芽，货物被限制在芽中。ESCRT-I和ESCRT-II定位于芽颈，并将ESCRT-0-泛素结构域募集到芽中。ESCRT-III亚基定位于芽颈，有效地裂解芽体形成腔内小泡。在该反应中产生的腔内小泡含有模型货物，但不含ESCRT。这些观察解释了ESCRT如何在反应中不被消耗的情况下直接从芽的细胞质一侧进行膜的萌发和断裂。 ESCRT循环的最后一步是AAA ATPase Vps4对ESCRT-III晶格的分解。Vps4与其辅因子Vta1一起组装在其膜结合的ESCRT-IIII底物上。测定了酵母Vta1的二聚体Vs1结构域与小ATPase和Vps4的b结构域的晶体结构。参与结构相互作用的残基是保守的，是体外结合和体内Cps1分选所必需的。对Vta1复合体与Vps4的下六角体环的复合体的建模表明，Vta1 VSL结构域的2倍轴平行于6倍轴的20度以内。这表明Vta1可能不会使Vps4的两个六聚体环交联，而是稳定了Vps4-Vta1复合体的阵列，用于ESCRT-III的分解。