MOLECULAR MECHANISMS OF MULTIVESICULAR BODY BIOGENESIS

摘要 内体系统将膜和来自质膜的蛋白质内化，然后在 在拓扑结构上不同的过程使膜内化，形成多囊泡体。 膜和相关物质内化到MVB是消化和降解MVB所必需的 溶酶体，也被专门的细胞用来产生外切体。一套十八个“E级” 从酵母到哺乳动物的保守蛋白质被认为调节并可能驱动 腔内小泡。这些蛋白质中的一部分或可能全部也被非裂解性病毒所使用，如艾滋病毒 从受感染细胞的质膜上发芽，这一过程在拓扑上等同于发芽 进入内体的管腔。对酵母和最近的哺乳动物系统的优雅研究为 洞察这些蛋白质是如何相互联系的，货物分子，以及内膜， 但基本上对这些或其他蛋白质如何驱动膜内陷和囊泡知之甚少。 放手。因为所需的膜曲率与传统的涂层囊泡驱动的膜曲率相反 内吞作用，很可能涉及到新的机制。我们发现使用速冻深蚀刻 电子显微镜下ESCRT-III蛋白与Snf7(哺乳动物系统中的hSnf7/CHMP4)相关 在质膜上组装成细丝，可以诱导形成紧密的圆形阵列和弯曲 细胞膜远离细胞质，在细胞表面形成芽并最终形成小管。我们 假设含有ESCRT-III的类似聚合物通常形成新生内体的颈部 囊泡，既限制囊泡的内容物，又在膜中引起必要的变形。我们的 计划是确定ESCRT-III聚合物的结构和动力学，在体外，在模型膜上，在 正常MVB生物发生的背景，以确定ESCRT-III如何组装和拆解 聚合物参与MVB的生物发生。目的1探讨ESCRT-III的同源和异源结构。 聚合物。目标2将定义调节ESCRT-III聚合物组装的机制。目标3将研究 AAA+ATPase Vps4对ESCRT-III聚合物的拆解最后，目标4将研究内生性 沉默前后受体下调过程中细胞中ESCRT-III蛋白的表达 Vps4和hSnf7蛋白的表达。

ABSTRACT The endosomal system internalizes membrane and protein from the plasma membrane, and then in a topologically distinct process internalizes membrane into itself to form multivesicular bodies (MVBs). Internalization of membrane and associated cargo into the MVB is essential for digestion and degradation in the lysosome, and is also used by specialized cells to generate exosomes. A set of eighteen "class E" proteins conserved from yeast to mammals is thought to regulate and probably drive the formation of intralumenal vesicles. Some or possibly all of these proteins are also used by nonlytic viruses such as HIV to bud from the plasma membrane of infected cells in a process that is topologically equivalent to budding into the lumen of the endosome. Elegant studies in yeast and more recently mammalian systems have lent insight into how these proteins associate with each other, cargo molecules, and the endosomal membrane, but essentially nothing is known about how these or other proteins drive membrane invagination and vesicle release. Because the required membrane curvature is opposite to that of traditional coated vesicle-driven endocytosis, it is likely that novel mechanisms are involved. We found using quick-freeze deep-etch electron microscopy that ESCRT-III proteins related to Snf7 (hSnf7/CHMP4 in mammalian systems) assemble into filaments on the plasma membrane that can be induced to form tight circular arrays and bend the membrane away from the cytoplasm, creating buds and eventually tubules on the cell surface. We hypothesize that similar ESCRT-III containing polymers normally create the neck of nascent endosomal vesicles, both confining the vesicle's contents and inducing the requisite deformation in the membrane. Our plan is to define the structure and dynamics of ESCRT-III polymers in vitro, on model membranes, and in the context of normal MVB biogenesis in order to determine how assembly and disassembly of ESCRT-III polymers participate in MVB biogenesis. Aim 1 will explore the structure of ESCRT-III homo- and hetero- polymers. Aim 2 will define mechanisms that regulate ESCRT-III polymer assembly. Aim 3 will study disassembly of ESCRT-III polymers by the AAA+ ATPase VPS4. Finally, Aim 4 will study endogenous ESCRT-III proteins in cells during the process of receptor downregulation before and after silencing expression of VPS4 and hSnf7 proteins.