CAREER: Regulation of cargo selection and ubiquitination by protein trafficking adaptors

为了应对不断变化的环境，细胞中的蛋白质被重新洗刷——特异性蛋白质从细胞表面移除，而其他蛋白质被选择性地靶向到表面。这种蛋白质重组被称为蛋白质运输，细胞必须做出正确的“决定”来控制蛋白质运输，确保最佳的细胞生长和存活。这项研究的目的是了解决定蛋白质运输的细胞决定是如何做出的。这个多学科项目将细胞和分子生物学与强大的新计算生物学方法相结合，以定义决定蛋白质如何选择性重新定位以响应环境变化的参数。调节这些细胞决定的关键角色是最近被描述的，但人们对其知之甚少的蛋白质家族，即α -阻滞蛋白。α -阻滞蛋白就像细胞的“邮差”，确保蛋白质在正确的时间被运送到正确的位置。尽管它们在蛋白质运输中起着重要的作用，但关于α -阻滞蛋白的功能仍然存在一些关键的尚未解决的问题，包括：1)α -阻滞蛋白如何识别它们所调节的蛋白质的离散子集？2)是什么因素控制着α -阻滞蛋白在何时何地与其转运的蛋白质相互作用？换句话说，这些分子邮差是如何知道哪些信要取，什么时候取，在哪里投递的呢？理解这些问题的答案至关重要；贩运决策过程中的缺陷会给细胞带来灾难性的后果。该研究项目的跨学科性质确保了各级学员——包括高中生、本科生和研究生——都能接触到广泛的科学方法。本项目的研究目标将整合到本科实验课程中，以确保本科研究人员在学习科学基础知识的同时体验科学发现的快感。还将设立大学生和高中生暑期实习，让学生在蛋白质运输的动态领域中为推进知识的边界做出贡献。这项研究的目标是用α -抑制因子（一种令人兴奋的新型转运适应因子）作为模型，定义控制选择性蛋白质转运的原则。α -阻滞蛋白保存于酵母和人类中，与具有良好特征和临床重要性的哺乳动物β -阻滞蛋白相关，是最近描述的一类转运适应子，在选择性蛋白质转运中起关键作用。虽然我们还没有认识到α -阻滞蛋白功能的广度，但在酵母中，它们与泛素连接酶Rsp5相互作用，以调节货物蛋白的运输命运。α -阻滞蛋白很少被研究，这里提出的实验将定义关键的翻译后调控和新的生物学功能，这些关键的蛋白质运输调节因子。具体而言，本研究将：1)定义α -抑制蛋白-Rsp5界面的破坏如何损害Rsp5泛素连接酶的效率；2)确定泛素化如何调节α -阻滞蛋白介导的转运，并确定Rsp5活性如何被限制以允许α -阻滞蛋白单泛素化；3)全面识别α -阻滞蛋白，并使用稳健的新计算方法定义指示α -阻滞蛋白-阻滞蛋白相互作用的基序，该方法采用进化特征来推断功能关系。该项目的结果将为如何管制贩运适应子、限制泛素偶联和实现选择性货物贩运提供新的范例。通过使用新颖的计算方法和互补的遗传和生化方法，本研究将建立管理贩运适配器功能的规则。转运适应因子决定了所有真核生物的货物定位，因此从这些实验中确定的“决策”规则将为具有不同兴趣的研究人员提供重要信息。

In response to a changing environment, proteins are reshuffled in the cell - specific proteins are removed from the cell surface while others are selectively targeted to the surface. This protein reshuffling is referred to as protein trafficking and cells must make the correct 'decisions' to control protein trafficking and ensure optimal cell growth and survival. The goal of this research is to understand how the cellular decisions that dictate protein trafficking are made. This multidisciplinary project pairs cell and molecular biology with powerful new computational biology approaches to define the parameters that determine how proteins are selectively relocalized in response to environmental changes. Key players in regulating these cellular decisions are a recently described, but poorly understood, family of proteins called the alpha-arrestins. Alpha-arrestins act as cellular 'mail carriers' ensuring that proteins are delivered to the right location at the right time. In spite of their important role in protein trafficking, there remain critical, yet unanswered questions about alpha-arrestin function including: 1) how do alpha-arrestins recognize the discreet subset of proteins they regulate? and 2) what factors control when and where alpha-arrestins interact with the proteins they traffic? In other words, how do these molecular mail carriers know which letters to pick up, when to pick them up and where to deliver them? Understanding the answers to these questions is essential; defects in the trafficking decision making process have catastrophic consequences for the cell. The interdisciplinary nature of this research project ensures that trainees at all levels - including high school, undergraduate and graduate students - gain exposure to a wide-array of scientific approaches. The research objectives of this project will be integrated into an undergraduate laboratory course to ensure that undergraduate researchers get to experience the thrill of scientific discovery while learning science fundamentals. Undergraduate and high school student summer internships will also be created to allow students to contribute to advancing the boundary of knowledge in the dynamic field of protein trafficking. The goal of the research is to define principles that govern selective protein trafficking using alpha-arrestins, an exciting new class of trafficking adaptor, as a model. The alpha-arrestins, conserved from yeast to humans and related to the well-characterized and clinically important mammalian beta-arrestins, are a recently described class of trafficking adaptor that play a critical role in selective protein trafficking. While we have yet to appreciate the breadth of alpha-arrestin function, in yeast they interact with the ubiquitin ligase Rsp5 to regulate the trafficking fate of cargo proteins. The alpha-arrestins are little studied and the experiments proposed here will define key posttranslational regulation of, and novel biological functions for, these critical regulators of protein trafficking. Specifically, this research will: 1) Define how disruption of the alpha-arrestin-Rsp5 interface impairs the ubiquitin ligase efficiency of Rsp5; 2) Determine how ubiquitination regulates alpha-arrestin-mediated trafficking and determine how Rsp5 activity is restricted to permit alpha-arrestin mono-ubiquitination; and 3) Comprehensively identify alpha-arrestin cargo proteins and define motifs that dictate alpha-arrestin-cargo interaction using a robust, new computational approach that employs evolutionary signatures to infer functional relationships. The results of this project will yield new paradigms for how trafficking adaptors are regulated, ubiquitin conjugation is restricted and selective cargo trafficking is achieved. Through the use of novel computational methods and complementary genetic and biochemical approaches, this research will establish the rules that govern trafficking adaptor function. Trafficking adaptors dictate cargo localization in all eukaryotes, thus the 'decision-making' rules identified from these experiments will provide vital information to researchers with diverse interests.