BERNSTEIN-DISSERTATION-2017.pdf (3.05 MB)
Identification and Characterization of Signal Motifs that Regulate Surface Levels of Membrane Proteins
thesisposted on 2018-02-08, 00:00 authored by Joshua David Bernstein
The correct trafficking of membrane bound proteins is essential for cellular function. Many diseases, arise from defects in cell surface trafficking of membrane proteins. The trafficking of membrane proteins is often mediated by the distinct signal motifs on the cargo proteins, however many proteins do not contain any of the established trafficking motifs. The continued identification and characterization of protein trafficking motifs will improve our understanding of cellular physiology and potentially lead to the development of therapeutics for protein trafficking diseases. Here, we demonstrate the utility of B31, a mutant S. cerevisiae strain lacking potassium (K+) efflux transporters, to study the regulation of functional trafficking of a reporter membrane protein (Kir2.1 channel) to the cell surface. B31 yeast heterologously expressing the Kir2.1 channel is unable to grow when cultured in high concentrations of extracellular K+. However, B31 expressing the Kir2.1 channel defective in post-Golgi trafficking or those fused with intracellular targeting motifs were rescued in high K+ media. Thus a growth rescue of B31 cells in high K+ media represents the reduction in the surface expression of the reporter Kir2.1 protein. Using this system, we have developed a method of screening a random C-terminal peptide library fused to Kir2.1 channel for signals which reduce the levels of Kir2.1 on the cell surface. Our screening system identified sequences that reduce the surface levels of proteins through multiple distinct trafficking pathways including endoplasmic reticulum (ER)-associated degradation, ER retrieval, clathrin-mediated endocytosis, and a ubiquitin ligase binding motif. Thus our system offers a unique tool to identify the trafficking motifs that regulate cell surface expression of membrane proteins.
DepartmentBiochemistry and Molecular Genetics
Degree GrantorUniversity of Illinois at Chicago