Natalie Hager - O'Donnell Lab
"Use of Fluorogen Activating Proteins for Quantitative Protein Trafficking Studies in Yeast"
Friday, March 26, 2021
12:10 PM
Virtual Zoom seminar
Abstract:
Recent advances in genetically encoded fluorescent probes have led to the development of fluorogen-activating proteins (FAPs). This technology has two components: a non-fluorescent single chain antibody (SCA), which is fused to a protein of interest, and a fluorogen, in this case a malachite-green (MG) dye derivative that is non-fluorescent on its own in solution. Binding of a fluorogen to a SCA results in a 20,000-fold fluorescence increase, producing a signal in the far-red range (633 nm) that is similar in intensity to enhanced Green Fluorescent Protein (eGFP). FAPs have distinct advantages over traditional fluorescent proteins that make them amenable to quantitative protein trafficking studies. Specifically, an impermeant MG derivative, MG-B-Tau, selectively detects cell surface proteins allowing for the measurement of protein internalization and recycling. Although developed in yeast, this technology had surprisingly not been used to detect localization in this model system until our recent work looking at the mammalian potassium channel, Kir21, at the cell surface. Using this system, we were able to identify a family of protein trafficking adaptors, the a-arrestin, as regulators of Kir2.1 trafficking. Since then, we: 1) optimized the SCA sequence for expression in yeast, 2) optimized imaging parameters for yeast, 3) created a series of FAP-tagged plasmids where a fusion protein can be expressed under a range of promoters, 4) generated a FAP-tagged suite of sub-cellular markers for in vivo co-localization studies, and 5) used FAPs to assess the endocytic trafficking of yeast membrane proteins Ste3. Thus, our work has demonstrated the powerful capabilities of this tool for protein trafficking studies in yeast. Additionally, using the FAP system we have further elucidated mechanisms and pathways needed for the a-arrestin-mediated trafficking of Kir2.1. In summary, we have utilized the FAP technology for our own studies in protein trafficking and have made this tool more readily available to the yeast community.