Ubiquitin-dependent sorting in endosomes and the TGN

项目摘要 该提案概述了一项综合战略，以深入了解3个相关流程 决定了细胞膜表面的完整性。许多细胞活动是 精确地依赖于质膜上各种蛋白质的水平和所有组成部分， 这反映了控制它们内化到内体中的运输途径的平衡， 它们从内体沿沿着循环路线转运回质膜， 在递送和在溶酶体中降解之前包装到内体管腔内囊泡中。 溶酶体降解膜蛋白的主要决定因素之一 是它们与泛素的共价连接，随后被一系列泛素识别， 分选受体确切地说，泛素化蛋白质是如何在核内体上聚集的 在结合到管腔内囊泡之前，膜的结构还不清楚。但我们 已经发现了一个小的4通道膜蛋白家族， 泛素化蛋白分离的亚结构域。在这里，我们将研究的性质 这些蛋白质，亚结构域的形成，以及货物和分选事件的类型， 这样的子域控制。 泛素也被证明是内化的信号，然而，确切的信号是： 通过许多多组分内化介导其识别的分选受体 设备.我们已经发现，网格蛋白包被囊泡的货物衔接子的一个家族， 内化以相对高的亲和力结合泛素。我们现在将决定这是否 家族，可能与其他Ub结合蛋白结合，作为一个关键组成部分， 细胞表面蛋白的泛素依赖性内化。 在降解蛋白质或允许其保留之间的关键排序决定之一 活性发生在早期的核内体，在那里蛋白质要么被包装成管状囊泡， 载体沿着循环途径到达质膜，或包装到腔内囊泡中 用于随后的递送和在溶酶体中的降解。我们最近完成了一个基因筛查 发现了从早期内体回收所需的大量新蛋白质机器。 这一过程通过Rag/Gtr GTP酶受到广泛的代谢控制， 独立于TORC 1。这里将继续这些观察与生物化学和遗传 实验将这种代谢调节与影响人类健康的运输机制联系起来， 蛋白质在循环系统中的运动。

Project Summary This proposal outlines an integrated strategy to gain insights into 3 related processes that determine the levels of integral membrane cell surface. Many cellular activities are exquisitely dependent on the level and repertoire of a variety proteins at the plasma membrane, which reflects a balance of trafficking pathways that control their internalization into endosomes, their transit from endosomes back to the plasma membrane along recycling routes, and their packaging into endosomal intraluminal vesicles prior to delivery and degradation in lysosomes. One of the main determinants for sending membrane proteins for lysosomal degradation is their covalent attachment to ubiquitin, that is subsequently recognized by a series of ubiquitin- sorting receptors. Exactly, how ubiquitinated proteins are physically clustered on endosomal membranes prior to incorporation into intraluminal vesicles has been unclear. However, we have discovered a family of small 4-pass membrane proteins that establish endosomal subdomains that ubiquitinated proteins segregate into. Here we will study the properties of these proteins, the formation of the subdomains, and the types of cargo and sorting events that such subdomains control. Ubiquitin has also been shown to be a signal for internalization, however, the exact sorting receptor(s) that mediate its recognition by many multi-component internalization apparatus. We have discovered that the one family of cargo adaptors for clathrin coated vesicle internalization binds ubiquitin with relatively high affinity. We will now determine whether this family, perhaps in combination with other Ub-binding proteins, serves as a critical component for ubiquitin-dependent internalization of cell surface proteins. One of the critical sorting decisions between degrading a protein or allowing it to remain active takes place in early endosomes, where proteins are either packaged into tubulo-vesicular carriers along a recycling route to the plasma membrane, or packaged into intraluminal vesicles for subsequent delivery and degradation in lysosomes. We recently completed a genetic screen that identified a plethora of new protein machinery required for recycling from early endosomes. This process is under broad metabolic control via the Rag/Gtr GTPases through a process that is independent of TORC1. Here will pursue these observations with biochemical and genetic experiments to connect this metabolic regulation with the trafficking machinery that effects protein movement through the recycling system.