ANALYSIS OF ESCRT FUNCTION IN ENDOLYSOSOMAL TRAFFICKING

运输所需的内体分选复合物（ESCRT）是一组相互作用的蛋白质， 负责内体内部的货物选择和腔内小泡的生物发生的复合物 多泡体，也称为晚期内体。进化保护和发现， ESCRT是拓扑学等价过程所必需的，包括病毒出芽，细胞动力学分裂， 和核膜闭合导致了现在被广泛接受的概念，即ESCRT是唯一参与 膜裂变的反应，共享这个拓扑结构。ESCRT-III蛋白通过改变构象起作用 聚合成膜重塑细丝，在内部螺旋-负弯曲- 膜表面的小管，最终拉动小管关闭驱动膜分裂和释放。 有趣的是，人体中有12种ESCRT-III蛋白在功能上是不可互换的。 除了鉴定不同的分子结合伴侣外，几乎没有结构上的区别。 来解释它们独特的生理重要性。我们最近做出了令人惊讶的 发现两种人类ESCRT-III蛋白-CHMP 1B和IST 1-组装成螺旋状的细丝 在膜小管的外正弯曲表面周围，在体外和体内形成外被膜， vivo.这种对正弯曲膜小管的意外偏好挑战了 与ESCRT-III丝相关的膜变形和裂变活动仅限于单一拓扑结构 并促使我们重新考虑ESCRT-III功能的既定范式。该项目将（1）定义 特定ESCRT-III均聚物和杂聚物聚合物的拓扑结构偏好以及相应的 内源性ESCRT-III蛋白在内体系统中的分布，（2）确定 在不同的内体货物运输途径中的代表性ESCRT-III蛋白，以及（3）比较 消耗不同ESCRT-III蛋白质对内体和溶酶体形态的影响。这项工作将 扩大我们对ESCRT-III膜重塑系统的理解， 内溶酶体系统内的运输和组织的未来研究。

The Endosomal Sorting Complex Required for Transport (ESCRT) machinery is a set of interacting protein complexes responsible for cargo selection and biogenesis of intralumenal vesicles inside endosomal multivesicular bodies, also known as late endosomes. Evolutionary conservation and the discovery that ESCRTs are required for topologically equivalent processes including viral budding, cytokinetic abscission, and nuclear envelope closure led to the now widely accepted concept that ESCRTs are uniquely involved in membrane fission for reactions that share this topology. ESCRT-III proteins act by changing conformation and polymerizing into membrane-remodeling filaments that spiral on the inside – negatively curved – surface of membrane tubules, ultimately pulling the tubules closed to drive membrane fission and release. Intriguingly, there are twelve ESCRT-III proteins in humans that are not functionally interchangeable. Beyond identification of different molecular binding partners, there has been little structural distinction among these proteins to explain their unique physiological importance. We recently made the surprising discovery that two human ESCRT-III proteins – CHMP1B and IST1 – assemble into filaments that spiral around the outside – positively curved – surface of membrane tubules, forming external coats in vitro and in vivo. This unexpected preference for positively curved membrane tubules challenges the dogma that the membrane deforming and fission activity associated with ESCRT-III filaments is limited to a single topology and prompts us to reconsider established paradigms for ESCRT-III function. This project will (1) define the topology preference of particular ESCRT-III homo- and heteropolymers polymers and the corresponding distribution of endogenous ESCRT-III proteins across the endosomal system, (2) establish the role(s) of representative ESCRT-III proteins in distinct endosomal cargo trafficking pathways, and (3) compare the effects of depleting different ESCRT-III proteins on endosomal and lysosomal morphology. This work will expand our understanding of the ESCRT-III membrane remodeling system with significant implications for future studies of trafficking and organization within the endolysosomal system.