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 这决定了细胞表面整体膜的水平。许多细胞活动都是 微妙地依赖于质膜上各种蛋白质的水平和谱带， 这反映了控制它们内化到内体的运输途径的平衡， 它们从内体沿循环途径返回到质膜，以及它们的 在运送和降解溶酶体之前，包装到内体腔内小泡中。 决定溶酶体降解的膜蛋白的主要因素之一 是它们与泛素的共价结合，随后被一系列泛素识别- 对受体进行分类。确切地说，泛素化的蛋白质是如何物理地聚集在内体上的 膜在被结合到腔内小泡之前一直不清楚。然而，我们 已经发现了一个小的四通膜蛋白家族，它可以建立内体 泛素化蛋白质所分离到的亚域。在这里，我们将研究的性质 这些蛋白质，亚区的形成，以及货物和分拣事件的类型 这样的子域控制着。 然而，泛素也被证明是内化的信号，然而，确切的 多组分内化介导其识别的分选受体(S) 仪器。我们已经发现，用于笼状蛋白涂层囊泡的一种货物适配器家族 内化以相对较高的亲和力结合泛素。我们现在要确定的是， 家族可能与其他Ub结合蛋白结合在一起，是 依赖泛素的细胞表面蛋白内化。 在降解蛋白质或允许其保留之间的关键分拣决定之一 活性发生在早期的内小体中，在那里蛋白质要么被包装成管泡 载体沿着循环途径到达质膜，或被包装成腔内小泡 用于随后的递送和溶酶体的降解。我们最近完成了一项基因筛查 这确定了从早期内吞体内回收所需的过多新的蛋白质机制。 这一过程通过RAG/GTR GTP酶受到广泛的代谢控制，通过一个过程 是独立于TORC1的。我们将用生物化学和遗传学来进行这些观察 将这种新陈代谢调节与影响 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.