Yeast Auxilin Regulation of Clathrin Dynamics

所有的真核细胞都通过一种称为内吞作用的过程从细胞外环境中摄取分子。 在最具特征的内吞过程中，一种称为网格蛋白的自组装蛋白在质膜内侧形成一种轻微内陷的网格状结构，称为网格蛋白包被的小坑。 当细胞外分子，如我们血液中的LDL颗粒，与其细胞表面受体结合时，复合物迁移到这些网格蛋白包被的凹坑之一中，并通过与网格蛋白接头蛋白与受体的相互作用被捕获。 在小窝捕获了一些具有结合货物的受体后，它进一步内陷到细胞中，并从质膜上夹断，形成网格蛋白包被的囊泡。 然后从网格蛋白包被的囊泡快速剥离网格蛋白包被，以允许网格蛋白再循环回到质膜，并允许裸囊泡与内体融合。网格蛋白循环的组装和拆卸阶段必须严格调节，以确保囊泡出芽和脱壳的有序进行，但如何在活细胞中调节这样的动态系统知之甚少。 本项目将使用芽殖酵母（酿酒酵母）的简易分子遗传学模型系统来定义生长素的体内功能，生长素是网格蛋白脱壳中Hsp70活性的关键调节剂。 酵母生长素（Swa2p）是一种由N端网格蛋白结合基序和泛素结合（乌巴）结构域、可用于与Hsp70相互作用的TPR结构域和可刺激Hsp70 ATP酶活性的C端J结构域组成的模块蛋白。 该项目将定义每个域对活细胞中生长素功能的贡献。更广泛的影响。除了了解网格蛋白动力学如何调节的科学重要性外，该项目将继续为科学教育和基础设施做出贡献。通过NSF职业奖发起的arf1合成致命筛选已由数百名本科生进行，使他们有机会参与科学的发现过程。此外，本科生已经克隆了几个在这个屏幕上发现的SWA基因。本科生的参与将继续让更多的学生参与以发现为基础的研究体验。

All eukaryotic cells take up molecules from the extracellular environment through a process called endocytosis. In the best-characterized endocytic process, a self-assembling protein called clathrin forms a slightly invaginated, lattice-like structure on the inside of the plasma membrane called a clathrin-coated pit. When an extracellular molecule, such as the LDL particles in our bloodstream, binds to its cell surface receptor, the complex migrates into one of these clathrin-coated pits and is captured through interaction with clathrin adaptor proteins with the receptor. After the pit has captured a few receptors with bound cargo, it invaginates further into the cell and pinches off from the plasma membrane to form a clathrin-coated vesicle. The clathrin coat is then rapidly stripped from the clathrin-coated vesicles to allow recycling of clathrin back to the plasma membrane, and to allow fusion the naked vesicle with an endosome. The assembly and disassembly phases of the clathrin cycle must be tightly regulated to ensure an orderly progression of vesicle budding and uncoating, but how such a dynamic system is regulated in living cells is poorly understood. This project will define the in vivo function of auxilin, a key regulator of Hsp70 activity in clathrin uncoating, using the facile molecular genetics of the budding yeast (Saccharomyces cerevisiae) for a model system. Yeast auxilin (called Swa2p) is a modular protein consisting of N-terminal clathrin binding motifs and a ubiquitin binding (UBA) domain, a centrally located TPR domain that may be used for interaction with Hsp70, and a C-terminal J domain that can stimulate the Hsp70 ATPase activity. This project will define the contribution of each domain to auxilin function in living cells.Broader Impacts.In addition to the scientific importance of understanding how clathrin dynamics is regulated, this project will continue to contribute to science education and infrastructure. Initiated through an NSF career award, the arf1 synthetic lethal screen has been performed by hundreds of undergraduate students, giving them the opportunity to participate in the discovery process of science. Moreover, undergraduate students have cloned several of the SWA genes discovered in this screen. The participation of undergraduate students will be continued to engage more students in a discovery-based research experience.