(2012). including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7. INTRODUCTION G proteinCcoupled receptors (GPCRs) are the most numerous and diverse superfamily of cell-surface receptors (Davenport (Burkholder and Hartwell, 1985 ; Nakayama [2013 ] and Alvaro and Thorner [2016 ]) that lead to activation of a mitogen/messenger-activated protein kinase whose actions result in cell–cycle arrest in the G1 phase, cause highly polarized growth (called shmoo formation) (Madden and Snyder, 1998 ), and induce the transcription of genes required to prepare a allele, it was reported that this polarization of the yeast pheromone receptor requires its internalization but not actin-dependent secretion (Suchkov is a pheromone-induced gene (Hartig light chain (Ig) of human immunoglobulin G (IgG) directs secretion (Szent-Gyorgyi open reading frame (ORF) that was also tagged in-frame at its C terminus with an octapeptide epitope (DYKDDDDK) from the Gene 10 protein of bacteriophage T7 (FLAG tag) and a (His)6 tract, which, as we demonstrated previously, do not alter any measurable function of this GDC0853 receptor (David on a plasmid, as well as a control expressing Ste2-FLAG-(His)6 from the same vector, were introduced into cells. Immunoblotting revealed that both FAP-containing proteins were expressed and, compared with the Ste2-FLAG-(His)6 control (Supplemental Figure S1B, left), exhibited the increase in size expected for these chimeric receptors (Supplemental Figure S1B, right). Thus, the human FAP sequences were no impediment to transcription and translation in yeast. However, reproducibly, the FAP2-Ste2 construct was expressed at a significantly higher level than FAP1-Ste2 (Supplemental Figure S1B, GDC0853 right). Moreover, when incubated briefly with their cognate fluorogens, only the cells expressing the FAP2-Ste2 construct yielded a readily detectable fluorescent signal and that fluorescence was located, as expected, largely at the cell periphery (Supplemental Figure S1C). To determine whether we could improve surface expression of FAP2-Ste2 while retaining the proper folding and function of both its FAP and receptor KLF11 antibody domains, the secretory signal sequences of three endogenous yeast proteins (MF1, Ste2, and Suc2) were installed, either in place of or immediately upstream of the Ig signal peptide (Supplemental Figure S2A), as described in detail in the Supplemental GDC0853 Material. Each of these different signal peptide constructs was integrated into the locus and expressed from the endogenous promoter. The MF1(1-83)-Ig-FAP2-Ste2 construct (see Supplemental Table S2 for full nucleotide sequence), which contains most of the prepro-leader sequence in the precursor of the GDC0853 secreted pheromone -factor (Fuller prefers to grow at somewhat acidic pH. Whether cells were propagated at a given pH and then incubated with fluorogen at the same pH (Figure 1B), or pregrown at pH 6.5 and then shifted to medium at a different pH and then incubated with fluorogen (unpublished data), stable labeling was observed only at values approaching pH 6. Therefore, in all subsequent experiments, cells were grown in medium buffered at pH 6.5. Examination of viable titer after exposing FAP-Ste2-expressing cells to fluorogen at pH 6.5 for 15 min at 30C demonstrated that exposure to the dye under these conditions had no toxic effect (Figure 1C). Open in a separate window FIGURE 1: Optimization of fluorogen binding to FAP-Ste2. (A) Cells (yAEA152) expressing FAP-Ste2 from the endogenous locus were grown to midCexponential phase in BSM, incubated with fluorogen (0.4 mM final concentration) either on ice without agitation or at 30C with agitation (1200 rpm) for the time periods indicated, washed and collected by brief centrifugation, and viewed by fluorescence microscopy (top panels) and bright field microscopy (bottom panels), as described under cells, even basal endocytosis of FAP-Ste2 GDC0853 was readily observable, which was, as expected, actin dependent because it was blocked by the presence of LatA (Figure 2C). Hence, in all subsequent experiments, we used cells expressing FAP-Ste2. Open in a separate window FIGURE 2: Absence of yapsins preserves full-length endocytosis-competent FAP-Ste2. (A) Strain DK102 ( 200 cells per sample) of A488-F or FAP-Ste2 at the cell periphery, relative to the starting intensity for each strain, quantified.