Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast, and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.

Cdc42-Specific GTPase-Activating Protein Rga1 Squelches Crosstalk between the High-Osmolarity Glycerol (HOG) and Mating Pheromone Response MAPK Pathways

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5′-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of “kinetic proofreading”. Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

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

The yeast mating pathway regulates haploid cell fusion to form diploids in response to pheromone signaling. Study of this pathway has led to important insights into the structure and function of mitogen-activated protein kinase (MAPK) cascades, yet our understanding of how external signals are converted into specific changes in gene expression and cell morphology is incomplete. For example, the regulators of directional growth (chemotropism) remain poorly defined. Upon pheromone exposure, yeast grow asymmetrically towards a nearby mating partner (chemotropic morphogenesis) and form a mating projection (shmoo). Using non-biased genome-wide screening, we identify >20 novel positive and negative regulators of pheromone gradient sensing, shmoo development, and mating. In addition to known regulators of exocytic and endocytic pathways, several are directly involved in translational control downstream of the G-protein-regulated pheromone and filamentous growth MAPK pathways. These include the Scp160 RNA-binding protein and ribosomal proteins Asc1, Rpl12b and Rpl19b. Importantly, pheromone treatment and Gα (Gpa1) activation both stimulate Scp160 binding to, and inhibition of, Asc1, which acts downstream of glucose-activated Gα (Gpa2) on the filamentous growth pathway. We also identify Rpl12b and Rpl19b as paralog-specific positive regulators of translation of specific mating pathway components, including Scp160. Thus, the different MAPK pathways converge at the level of translational control to regulate signaling.

MAP Kinase Regulation of theCandida albicansPheromone Pathway

ABSTRACTWe investigated the relationships of the Cek1 and Cek2 mitogen-activated protein (MAP) kinases and the putative MAP kinase phosphatase Cpp1 in the mating process ofCandida albicans. Mutants of theCPP1gene are hyperresponsive to pheromone, generating large halos, high levels of projections, and an increase in pheromone-responsive gene expression. Mating-type-homozygous opaque cells that lack both kinases are sterile, consistent with previous observations, although several lines of evidence show that the two kinases do not simply provide redundant functions in the mating process. Loss ofCEK1reduces mating significantly, to about 0.3% of wild-type strains, and also reduces projection formation and pheromone-mediated gene expression. In contrast, loss ofCEK2has less of an effect, reducing mating to approximately one-third that of the wild-type strain and moderately reducing projection formation but having little influence on the induction of gene expression. However, loss of Cek2 function reduces adaptation to pheromone-mediated arrest. The mutation enhances pheromone response halos to a level similar to that ofcpp1mutants, although thecpp1mutants are considerably more mating defective than thecek2mutant. The doublecek2 cpp1mutant shows enhanced responsiveness relative to either single mutant in terms of gene expression and halo formation, suggesting the kinase and phosphatase roles in the adaptation process are independent. Analysis of protein phosphorylation shows that Cek1 undergoes pheromone-mediated phosphorylation of the activation loop, and this phosphorylation is enhanced in cells lacking either the Cpp1 phosphatase or the Cek2 kinase. In addition, Cek1-GFP shows enhanced nuclear localization in response to pheromone treatment. In contrast, Cek2 shows no evidence for pheromone-mediated phosphorylation or pheromone-mediated nuclear localization. Intriguingly, however, deletion ofCPP1enhances both the phosphorylation state and the nuclear localization of Cek2-GFP. Overall, these results identify a complex interaction among the MAP kinases and MAP kinase phosphatase that function in theC. albicansmating pathway.IMPORTANCEMAP kinases and their regulators are critical components of eukaryotic signaling pathways implicated in normal cell behavior as well as abnormal behaviors linked to diseases such as cancer. The mating pathway of the yeastSaccharomycescerevisiaewas central in establishing the MAP kinase paradigm. Here we investigate the mating pathway in a different ascomycete, the fungal pathogenC. albicans. In this dimorphic fungus MAP kinases are also implicated in the mating response, with two MAP kinases apparently playing redundant roles in the mating process. This work establishes that while some level of mating can occur in the presence of a single kinase, the Cek1 kinase is most important for mating, while the Cek2 kinase is involved in adaptation to signaling. While both kinases appear to be themselves regulated by dephosphorylation through the action of the Cpp1 phosphatase, this process appears important for mating only in the case of Cek1.

Timing of gene expression in a cell-fate decision system

During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating-responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contribute to determine the kinetics of transcription in a simplified cell-fate decision system.One Sentence SummaryQuantitative and dynamic single cell measurements in the yeast mating pathway uncover a complex temporal orchestration of gene expression events.

Protein expression patterns of the yeast mating response

We systematically studied dynamic protein expression patterns of the mating pathway under two conditions with high temporal resolution.