MOLECULAR MECHANISMS OF MULTIVESICULAR BODY BIOGENESIS

摘要 内体系统从质膜内化膜和蛋白质，然后在一个 拓扑上不同的过程将膜内化到自身中以形成多泡体（MVB）。 膜和相关货物内化到MVB中对于消化和降解是必需的， 溶酶体，也被专门的细胞用来产生外泌体。一套十八个“E类” 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。这些蛋白质中的一些或可能全部也被非裂解性病毒（如HIV）使用 从受感染细胞的质膜上出芽，其过程在拓扑学上等同于出芽 进入内体的内腔。在酵母和最近的哺乳动物系统中进行的优雅研究， 深入了解这些蛋白质如何相互关联，货物分子，和内体膜， 但对于这些或其他蛋白质如何驱动膜内陷和囊泡， release.由于所需的膜曲率与传统的包被囊泡驱动的膜曲率相反， 内吞作用，很可能涉及新的机制。我们发现使用速冻深蚀刻 电子显微镜下，ESCRT-III蛋白与Snf 7（哺乳动物系统中的hSnf 7/CHMP 4）相关 在质膜上组装成细丝，这些细丝可以被诱导形成紧密的圆形阵列并弯曲 细胞膜脱离细胞质，在细胞表面形成芽和小管。我们 假设类似的含有ESCRT-III的聚合物通常产生新生内体的颈 囊泡，既限制囊泡的内容物，又引起膜的必要变形。我们 计划是在体外、模型膜上和体内确定ESCRT-III聚合物的结构和动力学。 正常MVB生物发生的背景，以确定ESCRT-III的组装和拆卸 聚合物参与MVB生物发生。目的1探索ESCRT-Ⅲ的同源和异源结构， 聚合物目的2将定义调节ESCRT-III聚合物组装的机制。目标3将研究 通过AAA+ ATP酶VPS 4分解ESCRT-III聚合物。最后，目标4将研究内源性 ESCRT-III蛋白在细胞受体下调过程中的沉默前后 VPS 4和hSnf 7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.