NSF Award
1553143
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. <br/><br/>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.
