Cargo Sorting and Intralumenal Vesicle Budding by the ESCRT Complexes

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蛋白直接结合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。 2007财政年度的进展 从酵母到人类保守的转运蛋白I所需的内体分选复合物（ESCRT-I）复合物指导泛素化跨膜蛋白的溶酶体降解和HIV病毒的出芽。酵母ESCRT-I含有Vps 23、Vps 28、Vps 37和Mvb 12四个亚基。我们确定了异源四聚体ESCRT-I复合物的晶体结构，其揭示了1：1：1：1亚基化学计量的高度不对称复合物。核心复合物长约18 nm，由一个连接到13 nm茎的头部分组成。与Elizabeth Conibear（不列颠哥伦比亚省大学）合作进行的功能研究表明，茎对于ESCRT-I的货物分类很重要，并被提议作为调节货物和其他ESCRT组件正确处置的间隔区。与Rodolfo Ghirdek（NIDDK）合作获得的流体动力学约束和我们自己实验室和其他实验室的晶体结构被用于生成完整的ESCRT-I溶液模型。结果显示ESCRT-I如何使用刚性茎和柔性系链的组合与脂质，货物和其他ESCRT复合物在25 nm的跨度上相互作用。这些概念使用结合研究来测试，以模型内体膜含有PI（3）P和通过将泛素-Cps 1（Cps 1是酵母ESCRT的正常货物）融合蛋白共价连接到脂质而产生的模型货物。

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. 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. Progress in FY2007 The Endosomal Sorting Complex Required for Transport-I (ESCRT-I) complex, which is conserved from yeast to humans, directs the lysosomal degradation of ubiquitinated transmembrane proteins and the budding of the HIV virus. Yeast ESCRT-I contains four subunits, Vps23, Vps28, Vps37 and Mvb12. We determined the crystal structure of the heterotetrameric ESCRT-I complex, which revealed a highly asymmetric complex of 1:1:1:1 subunit stoichiometry. The core complex is nearly 18 nm long, and consists of a headpiece attached to a 13 nm stalk. Functional studies carried out in collaboration with Elizabeth Conibear (Univ. of British Columbia) showed that the stalk is important for cargo sorting by ESCRT-I, and is proposed to serve as a spacer regulating the correct disposition of cargo and other ESCRT components. Hydrodynamic constraints obtained in collaboration with Rodolfo Ghirlando (NIDDK) and crystallographic structures from our own lab and from others were used to generate a model of intact ESCRT-I in solution. The results show how ESCRT-I uses a combination of a rigid stalk and flexible tethers to interact with lipids, cargo, and other ESCRT complexes over a span of 25 nm. These concepts were tested using binding studies to model endosomal membranes containing PI(3)P and a model cargo generated by covalently linking ubiquitin-Cps1 (Cps1 is a normal cargo of the yeast ESCRTs) fusion protein to lipids.