Investigation of the mechanisms of endocytic protein recruitment

 描述（由申请方提供）：网格蛋白介导的内吞作用（CME）是一种保守的过程，负责营养物质和质膜组分的选择性摄取。CME是细胞与外部环境相互作用并对其做出反应的重要方式。异常的CME与许多疾病状态有关，包括动脉粥样硬化和糖尿病。此外，许多病毒通过内吞作用进入细胞，内吞作用的缺陷通过影响细胞极性、增殖和迁移而有助于肿瘤的转移潜力。因此，了解内吞作用背后的机制对于了解正常细胞功能和发病机制至关重要。 芽殖酵母Saccharomycescerevisiae具有易于遗传操作、基因冗余度低、仅存在一条胞吞途径等优点，是研究CME的理想体系。酵母中的CME涉及约60种内吞蛋白的有序膜募集、活性和分解。以前在酵母中的工作已经建立了每种蛋白质在质膜上出现的精确顺序，几乎所有这些信息都被证明可以直接应用于更复杂的生物体。然而，这些蛋白质中的许多被招募到质膜的机制，特别是在内吞位点起始期间，仍然知之甚少。本研究的目的是确定内吞膜蛋白是如何被募集到质膜上的。具体来说，这项工作将定义Ede 1泛素化在下游蛋白质募集中的作用，并将鉴定将Las17募集到质膜的蛋白质。 肌动蛋白尾部组装可以在微珠和合成膜上重构。扩展这项工作，内吞作用将重建与Ede 1，一个内吞蛋白的网站启动关键包被的合成膜。将Ede 1包被的膜置于表达标记的内吞蛋白的酵母细胞提取物中。用于确认成功重建的主要结局将包括胞吞蛋白募集至膜、肌动蛋白尾形成和囊泡释放。为了检验Ede 1泛素化影响下游蛋白质募集的假设，将使用质谱法将蛋白质募集与含有Ede 1或泛素化Ede 1的膜进行比较。为了检验至少一种上游蛋白质是用于Las17募集的支架的假设，将使用上述体外测定和活细胞中的创新策略。一种早期（Sla2）和晚期（Sla3）之间的融合蛋白， （Las17）内吞蛋白将在酵母中表达。这种构建体有望拯救las17敲除细胞，并消除对主要用作支架的中间蛋白的需求。这两种方法有望协同发展，以确定参与内吞位点启动和稳定的机制，从而增加我们对CME的理解。

 DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis (CME) is a conserved process responsible for the selective uptake of nutrients and plasma membrane components. CME is a crucial way for cells to interact with, and react to, their external environments. Aberrant CME is implicated in many disease states, including atherosclerosis and diabetes. Additionally, a number of viruses enter the cell by endocytosis, and defects in endocytosis contribute to the metastatic potential of tumors by affecting cell polarity, proliferation, and migration. Understanding the mechanisms behind endocytosis is thus crucial for understanding normal cell function and pathogenesis. The budding yeast Saccharomyces cerevisiae makes an ideal system for studying CME, due to advantages such as easy genetic manipulation, low gene redundancy, and the existence of only one major endocytosis pathway. CME in yeast involves the ordered membrane recruitment, activity, and disassembly of ~60 endocytic proteins. Previous work in yeast has established the precise order of each protein's appearance at the plasma membrane, and almost all of this information has proven directly applicable to more complex organisms. However, the mechanism by which many of these proteins are recruited to the plasma membrane, particularly during endocytic site initiation, is still poorly understood. The goal of this research is to determine how endocytic membrane proteins are recruited to the plasma membrane. Specifically, this work will define the role of Ede1 ubiquitination in downstream protein recruitment and will identify proteins that recruit Las17 to the plasma membrane. Actin tail assembly can be reconstituted on microbeads and synthetic membranes. Extending this work, endocytosis will be reconstituted on synthetic membranes coated with Ede1, an endocytic protein critical for site initiation. Ede1-coated membranes will be placed in cell extracts from yeast expressing tagged endocytic proteins. Primary outcomes used to confirm successful reconstitution will include endocytic protein recruitment to membranes, actin tail formation, and vesicle release. To test the hypothesis that Ede1 ubiquitination effects downstream protein recruitment, mass spectrometry will be used to compare protein recruitment to membranes containing either Ede1 or ubiquitinated Ede1. To test the hypothesis that at least one upstream protein is a scaffold for Las17 recruitment, the above in vitro assay and an innovative strategy in live cells will be used. A fusion protein between an early (Sla2) and a late (Las17) endocytic protein will be expressed in yeast. This construct is expected to rescue las17 knockout cells and obviate the need for intermediate proteins that serve primarily as scaffolds. These two approaches promise to develop synergistically to identify mechanisms involved in endocytic site initiation and stabilization and thus increase our understanding of CME.