ANALYSIS OF ESCRT FUNCTION IN ENDOLYSOSOMAL TRAFFICKING

运输所需的内体分选复合体(ESCRT)是一组相互作用的蛋白质 负责选择货物和腔内小泡在内体内的生物发生的复合体 多囊泡小体，又称晚期内体。进化守恒和发现 ESCRT是拓扑上等价的过程所必需的，包括病毒发芽、细胞分裂、 和核包膜关闭导致了现在被广泛接受的概念，ESCRT是唯一参与其中的 具有这种拓扑结构的反应的膜裂变。ESCRT-III蛋白通过改变构象发挥作用 并聚合成膜重塑的细丝，这些细丝在内部螺旋-负弯曲- 膜表面的小管，最终拉近小管，以驱动膜分裂和释放。 有趣的是，在人类中有12种ESCRT-III蛋白在功能上是不可互换的。 除了识别不同的分子结合伙伴外，几乎没有结构上的区别 在这些蛋白质中解释它们独特的生理重要性。我们最近做了一个令人惊讶的事情 发现两种人类ESCRT-III蛋白-CHMP1B和IST1-组装成螺旋状的细丝 围绕膜小管的外正曲面，在体外和体外形成外膜。 活着。这种对正弯曲膜小管的意外偏好挑战了这样一种教条，即 与ESCRT-III细丝相关的膜变形和分裂活动限于单一拓扑结构 并促使我们重新考虑ESCRT-III功能的既定范例。该项目将(1)定义 特定ESCRT-III均聚和杂聚聚合物的拓扑择优及相应的 内源性ESCRT-III蛋白在整个内体系统中的分布，(2)确定eSCRT-III的作用(S) 有代表性的ESCRT-III蛋白在不同的内体货物运输途径中，以及(3)比较 去除不同ESCRT-III蛋白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，对 关于内溶酶体系统内的贩运和组织的未来研究。

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.