| Title: | Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging |
| Author(s): | Emmerstorfer-Augustin A; Augustin CM; Shams S; Thorner J; |
| Address: | "Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202. Department of Mechanical Engineering, University of California, Berkeley, Berkeley, CA 94720-3202" |
| ISSN/ISBN: | 1939-4586 (Electronic) 1059-1524 (Print) 1059-1524 (Linking) |
| Abstract: | "To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7" |
| Keywords: | "Cell Membrane/metabolism Down-Regulation *Endocytosis Fluorescent Dyes/*metabolism Ligands Receptors, Mating Factor/*metabolism Recombinant Fusion Proteins/*metabolism Reproducibility of Results Saccharomyces cerevisiae/*cytology/*metabolism Saccharomyces;" |
| Notes: | "MedlineEmmerstorfer-Augustin, Anita Augustin, Christoph M Shams, Shadi Thorner, Jeremy eng R01 GM021841/GM/NIGMS NIH HHS/ S10 OD018136/OD/NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2018/09/13 Mol Biol Cell. 2018 Nov 1; 29(22):2720-2736. doi: 10.1091/mbc.E18-07-0424. Epub 2018 Sep 12" |
