Yeast Pheromone Response Pathway Research Articles

Screening microbially produced Δ9-tetrahydrocannabinol using a yeast biosensor workflow

Microbial production of cannabinoids promises to provide a consistent, cheaper, and more sustainable supply of these important therapeutic molecules. However, scaling production to compete with traditional plant-based sources is challenging. Our ability to make strain variants greatly exceeds our capacity to screen and identify high producers, creating a bottleneck in metabolic engineering efforts. Here, we present a yeast-based biosensor for detecting microbially produced Δ9-tetrahydrocannabinol (THC) to increase throughput and lower the cost of screening. We port five human cannabinoid G protein-coupled receptors (GPCRs) into yeast, showing the cannabinoid type 2 receptor, CB2R, can couple to the yeast pheromone response pathway and report on the concentration of a variety of cannabinoids over a wide dynamic and operational range. We demonstrate that our cannabinoid biosensor can detect THC from microbial cell culture and use this as a tool for measuring relative production yields from a library of Δ9-tetrahydrocannabinol acid synthase (THCAS) mutants.

MAPK modulation of yeast pheromone signaling output and the role of phosphorylation sites in the scaffold protein Ste5.

Mitogen-activated protein kinases (MAPKs) mediate numerous eukaryotic signaling responses. They also can modulate their own signaling output via positive or negative feedback loops. In the yeast pheromone response pathway, the MAPK Fus3 triggers negative feedback that dampens its own activity. One target of this feedback is Ste5, a scaffold protein that promotes Fus3 activation. Binding of Fus3 to a docking motif (D motif) in Ste5 causes signal dampening, which was proposed to involve a central cluster of phosphorylation sites in Ste5. Here, we reanalyzed the role of these central sites. Contrary to prior claims, phosphorylation-mimicking mutations at these sites did not impair signaling. Also, the hyperactive signaling previously observed when these sites were mutated to nonphosphorylatable residues arose from their replacement with valine residues and was not observed with other substitutes. Instead, a cluster of N-terminal sites in Ste5, not the central sites, is required for the rapid dampening of initial responses. Further results suggest that the role of the Fus3 D motif is most simply explained by a tethering effect that promotes Ste5 phosphorylation, rather than an allosteric effect proposed to regulate Fus3 activity. These findings substantially revise our understanding of how MAPK feedback attenuates scaffold-mediated signaling in this model pathway.

Real-Time Genetic Compensation Defines the Dynamic Demands of Feedback Control

Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments can often identify the existence of feedback interactions but are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed-loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors, in real time, the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver-on the fly-an optogenetically enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.

Mathematical modeling reveals differential regulation of MAPK activity by phosphatase proteins in the yeast pheromone response pathway.

To prevent indefinite cellular responses to external signals, cells utilize various adaptation mechanisms. The yeast mating-response pathway is a model cellular system that exhibits adaptation to persistent external signals. This pathway employs a mitogen-activated protein kinase (MAPK) cascade which is composed of two well-known negative feedback inhibitions that involve the yeast phosphatase proteins Ptp3 and Msg5. The phosphorylated form of the yeast MAPK protein Fus3 (pFus3) triggers the phosphorylation of both phosphatases, but transcriptionally upregulates only Msg5. To study the biological rationale for the existence of two distinct negative feedback inhibitions acting on pFus3, we used published experimental data to develop a mathematical model which quantifies the inhibitory roles of these phosphatase proteins on pFus3. Our analyses show that the inhibition of pFus3 due to Ptp3 is largely independent of the signal profile, and is most impactful at early time points after pheromone induction. Conversely, the feedback inhibition due to Msg5 is highly dependent on the signal profile, and is most influential after pFus3 attains its maximum cellular abundance. Similarly, Ptp3 reduces the variation in the pFus3 dynamics at early time points while the noise-reduction effects of Msg5 become stronger as time passes.

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding.

Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes.

The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK) pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS), and barcode analysis by sequencing (Bar-Seq). Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported), including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating.

Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant

Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their invivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an invivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

Information Flow in Interaction Networks II: Channels, Path Lengths, and Potentials

In our previous publication, a framework for information flow in interaction networks based on random walks with damping was formulated with two fundamental modes: emitting and absorbing. While many other network analysis methods based on random walks or equivalent notions have been developed before and after our earlier work, one can show that they can all be mapped to one of the two modes. In addition to these two fundamental modes, a major strength of our earlier formalism was its accommodation of context-specific directed information flow that yielded plausible and meaningful biological interpretation of protein functions and pathways. However, the directed flow from origins to destinations was induced via a potential function that was heuristic. Here, with a theoretically sound approach called the channel mode, we extend our earlier work for directed information flow. This is achieved by constructing a potential function facilitating a purely probabilistic interpretation of the channel mode. For each network node, the channel mode combines the solutions of emitting and absorbing modes in the same context, producing what we call a channel tensor. The entries of the channel tensor at each node can be interpreted as the amount of flow passing through that node from an origin to a destination. Similarly to our earlier model, the channel mode encompasses damping as a free parameter that controls the locality of information flow. Through examples involving the yeast pheromone response pathway, we illustrate the versatility and stability of our new framework.

Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.

The Ankyrin Repeats and DHHC S-acyl Transferase Domain of AKR1 Act Independently to Regulate Switching from Vegetative to Mating States in Yeast

Signal transduction from G-protein coupled receptors to MAPK cascades through heterotrimeric G-proteins has been described for many eukaryotic systems. One of the best-characterised examples is the yeast pheromone response pathway, which is negatively regulated by AKR1. AKR1-like proteins are present in all eukaryotes and contain a DHHC domain and six ankyrin repeats. Whilst the DHHC domain dependant S-acyl transferase (palmitoyl transferase) function of AKR1 is well documented it is not known whether the ankyrin repeats are also required for this activity. Here we show that the ankyrin repeats of AKR1 are required for full suppression of the yeast pheromone response pathway, by sequestration of the Gβγ dimer, and act independently of AKR1 S-acylation function. Importantly, the functions provided by the AKR1 ankyrin repeats and DHHC domain are not required on the same molecule to fully restore WT phenotypes and function. We also show that AKR1 molecules are S-acylated at locations other than the DHHC cysteine, increasing the abundance of AKR1 in the cell. Our results have important consequences for studies of AKR1 function, including recent attempts to characterise S-acylation enzymology and kinetics. Proteins similar to AKR1 are found in all eukaryotes and our results have broad implications for future work on these proteins and the control of switching between Gβγ regulated pathways.

Signal response sensitivity in the yeast mitogen-activated protein kinase cascade.

The yeast pheromone response pathway is a canonical three-step mitogen activated protein kinase (MAPK) cascade which requires a scaffold protein for proper signal transduction. Recent experimental studies into the role the scaffold plays in modulating the character of the transduced signal, show that the presence of the scaffold increases the biphasic nature of the signal response. This runs contrary to prior theoretical investigations into how scaffolds function. We describe a mathematical model of the yeast MAPK cascade specifically designed to capture the experimental conditions and results of these empirical studies. We demonstrate how the system can exhibit either graded or ultrasensitive (biphasic) response dynamics based on the binding kinetics of enzymes to the scaffold. At the basis of our theory is an analytical result that weak interactions make the response biphasic while tight interactions lead to a graded response. We then show via an analysis of the kinetic binding rate constants how the results of experimental manipulations, modeled as changes to certain of these binding constants, lead to predictions of pathway output consistent with experimental observations. We demonstrate how the results of these experimental manipulations are consistent within the framework of our theoretical treatment of this scaffold-dependent MAPK cascades, and how future efforts in this style of systems biology can be used to interpret the results of other signal transduction observations.

Systems Biology: A Switch for Sex

The yeast pheromone response pathway works in a graded fashion, such that more pheromone leads to more response, but a recent study has shown that small modifications convert it into a bistable switch, with implications for the evolution and engineering of reaction networks.

Differential effects of RGS proteins on Gα q and Gα 11 activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human Gα q and Gα 11 spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of Gα-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both Gα q and Gα 11 but, in contrast, RGS5 and RGS16 were much less effective against Gα 11 than Gα q. Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active Gα q QL/Gα 11 QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-Gα specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of Gα q/Gα 11 enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both Gα subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential Gα activity, further highlighting that RGS proteins can discriminate between two very closely related Gα subunits.

Investigating RGS proteins in yeast

Regulator of G protein signalling (RGS) proteins are vital in the adaptation of cells to stimulation via G protein-coupled receptors. Yeast have been integral in elucidating the important role that RGS proteins play within cellular processes. In addition to extensive characterisation of the endogenous RGS proteins, these organisms have enabled the identification and analysis of numerous mammalian homologues. The simplicity and plasticity of the yeast pheromone-response pathway has facilitated studies which would have been impossible in mammalian systems and it is certain that yeast will continue to have a great impact on this field of research in the future.

Regulated cell-to-cell variation in a cell-fate decision system

Here we studied the quantitative behaviour and cell-to-cell variability of a prototypical eukaryotic cell-fate decision system, the mating pheromone response pathway in yeast. We dissected and measured sources of variation in system output, analysing thousands of individual, genetically identical cells. Only a small proportion of total cell-to-cell variation is caused by random fluctuations in gene transcription and translation during the response ('expression noise'). Instead, variation is dominated by differences in the capacity of individual cells to transmit signals through the pathway ('pathway capacity') and to express proteins from genes ('expression capacity'). Cells with high expression capacity express proteins at a higher rate and increase in volume more rapidly. Our results identify two mechanisms that regulate cell-to-cell variation in pathway capacity. First, the MAP kinase Fus3 suppresses variation at high pheromone levels, while the MAP kinase Kss1 enhances variation at low pheromone levels. Second, pathway capacity and expression capacity are negatively correlated, suggesting a compensatory mechanism that allows cells to respond more precisely to pheromone in the presence of a large variation in expression capacity.

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Mathematical Modeling of RGS and G-Protein Regulation in Yeast

G-protein-activated signaling pathways are capable of adapting to a persistent external stimulus. Desensitization is thought to occur at the receptor level as well as through negative feedback by a family of proteins called regulators of G-protein signaling (RGS). The pheromone response pathway in yeast is a typical example of such a system, and the relative simplicity of this pathway makes it an attractive system in investigating the regulatory role of RGS proteins. Two studies have used computational modeling to gain insight into how this pathway is regulated (Hao et al., 2003; Yi et al., 2003). This article provides an introduction to computational analysis of signaling pathways by developing a mathematical model of the pheromone response pathway that synthesizes the results of these two investigations. Our model qualitatively captures many features of the pathway and suggests an additional mechanism for pathway inactivation. It also illustrates that a complete understanding of signaling pathways requires an investigation of their time-dependent behavior.

Yeast-Based Screening for Inhibitors of RGS Proteins

This article provides information on two screening platforms for the identification of regulators of G-protein signaling (RGS) protein modulators. Utilization of the yeast pheromone response pathway enabled the creation of a functional screen for RGS4 modulators. The RGSZ1-focused screen employs advances in yeast two-hybrid screening technologies and targets the protein-protein interface of the RGS domain/Galpha interaction. Moreover, the RGSZ1 screen provides the opportunity to multiplex the screening of two targets of interest, given the development of two different luciferase reporter genes that enabled sequential determination and intraassay controls. The screen formats were validated, implemented, and conducted as automated 384-well, liquid-based, high-throughput small molecule screens. Primary "hits" were confirmed using benchtop 96-well formats of these assays and advanced to in vitro functional evaluation assays. The yeast-based assay platforms provide robust cellular assays that result in the identification of small molecule modulators for both RGS targets. These molecules can serve both as tools with which to probe biological implications of RGS proteins and as potential starting points toward the development of novel modulators of G-protein signaling pathways. Such modulators may show potential for controlling and treating diseases resulting from inappropriate activity of G-protein signaling pathways.

Engineered Yeasts as Reporter Systems for Odorant Detection

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand‐dependent signaling of heterologous expressed G‐protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Gα proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Gα protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand‐induced expression of a FUS1‐HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand‐dependent expression of a FUS1‐Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat I7 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 × 10−8 to 5 × 10−6 M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat I7 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.

Identification of a novel mitogen-activated protein kinase kinase activation domain recognized by the inhibitor PD 184352.

Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 micro M). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding.