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

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.

Molecular mechanisms of chemotropism and cell fusion in unicellular fungi

“…Because the localization of the receptor to the shmoo tip does not require actin-based transport, but requires its endocytic removal, Suchkov et al (2010) proposed that receptor polarization is achieved by a mechanism blocking receptor internalization at the cell front. However, recent investigations using fluorogen-activating protein (FAP) tagging to specifically visualize the fate of surface-localized receptor concluded that no region of the plasma membrane is immune to internalization (Emmerstorfer-Augustin et al, 2018). This suggests that receptor accumulation at the shmoo tip does not rely on protection from endocytosis but on de novo production and secretion, which may be polarized independently from actin-based transport as observed in other instances (Bendezú and Martin, 2011;Yamamoto et al, 2010).…”

“…Accordingly, in S. cerevisiae, Ste2 (the α-factor receptor expressed in a-cells) is constitutively expressed and present over the entire cell surface in mitotically proliferating cells. However, pheromones trigger the rapid endocytosis of Ste2 with a halftime of ∼6 min (Emmerstorfer-Augustin et al, 2018), which then becomes enriched at the shmoo (the mating projection) cortex ∼30 min later. This observation poses a significant challenge to the idea of spatial decoding, as even at this late stage, the cell is able to start a new site of growth if the gradient direction changes (Moore et al, 2013).…”

Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis

“…In conclusion, as more examples of the application of FAP-based pH sensors to measure and analyze the endocytosis of various cargo in single cells are documented (e.g. Emmerstorfer-Augustin et al, 2018; Holleran et al, 2013; Perkins et al, 2018a; Perkins et al, 2018b; Pratt et al, 2017), the feasibility and utility of the high-throughput method described here for systems biology studies of the endocytic trafficking of different, endogenous transmembrane proteins, and especially, ligand-independent transporters, channels and other cargoes, will become increasingly important.…”

Translational control as a novel regulator of gradient sensing and chemotropism in yeast

“…However, their impact upon the angle of incidence between mating partners (Figure 4C) suggests they also intimately affect gradient sensing. Since pheromone signaling rearranges the spatial distribution of the Ste2 pheromone receptor from the entire cell surface to the shmoo tip 76 , these mutants likely impinge upon receptor cycling and, hence, local signaling. This may also involve localization of a mobile polarity site (or patch) that serves as a site of communication where pheromones are released and sensed 77,78 .…”