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一个基本过程。内吞作用、细胞内运输和分泌囊泡的形成以及线粒体分裂都是向内出芽的例子。在网格蛋白介导的胞吞作用的经典例子中，胞质蛋白动力蛋白在膜颈外形成阵列，膜裂变是由GTP的水解热力学驱动的。多泡体（MVBs）的形成是向外出芽的典型例子。MVBs是在注定与溶酶体融合的核内体成熟过程中形成的，并介导泛素化膜蛋白向溶酶体的分选。内核体的部分限制膜内化形成腔内囊泡（ILVs）。当MVB与溶酶体融合时，ILV含量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。包膜病毒从质膜出芽和细胞分裂（细胞质分裂）是外出芽事件的其他例子。MVB形成过程中的外出芽、病毒出芽和细胞质分裂都是由细胞质导向的。由于细胞质溶胶与新生芽颈的内部而不是外部接触，膜裂变的机制与向内出芽有根本的不同，并且利用了完全不同的蛋白质机制。在酵母中发现了负责MVB形成的ESCRT机制，这是了解向外出芽的一个重大突破。ESCRT机制在整个真核生物中是保守的，哺乳动物的许多包膜病毒使用ESCRT途径发芽，包括HIV-1。胞质分裂中膜颈的闭合也使用ESCRT途径。

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.