Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging

TL;DR

This study introduces a fluorogen-activating protein (FAP) tagging method to visualize yeast pheromone receptor Ste2 endocytosis in live cells, revealing new insights into its internalization mechanisms and the roles of specific endocytic adaptors.

Contribution

The paper demonstrates the first use of FAP tagging for studying integral membrane protein trafficking in yeast, providing a brighter, more distinct visualization of receptor internalization.

Findings

FAP tagging yields brighter plasma membrane signals than GFP or mCherry.

FAP-Ste2 internalization involves specific endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.

Deletion of GPI-anchored proteases was necessary for optimal FAP-Ste2 expression.

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.