Golf complements a GPA1 null mutation in Saccharomyces cerevisiae and functionally couples to the STE2 pheromone receptor

We have produced a plasmid designed for the expression of heterologous G protein alpha subunits in the yeast Saccharomyces cerevisiae. Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein alpha subunit, to the yeast pheromone response pathway. The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal1- null mutation. A similar response is obtained when an alternative G protein alpha subunit, G(olf), is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.     

Engineered Yeasts as Reporter Systems for Odorant Detection

Abstract

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.     

Regulation of the yeast pheromone response pathway by G protein subunits.

The yeast GPA1, STE4, and STE18 genes encode proteins homologous to the respective alpha, beta and gamma subunits of the mammalian G protein complex which appears to mediate the response to mating pheromones. Overexpression of the STE4 protein by the galactose-inducible GAL1 promoter caused activation of the pheromone response pathway which resulted in cell-cycle arrest in late G1 phase and induction of the FUS1 gene expression, thereby suppressing the sterility of the receptor-less mutant delta ste2. Disruption of STE18, in turn, suppressed activation of the pheromone response induced by overexpression of STE4, suggesting that the STE18 product is required for the STE4 action. However, overexpression of both the STE4 and STE18 proteins did not generate a stronger pheromone response than overexpression of STE4 in the presence of wild-type levels of STE18. These results suggest that the beta subunit is the limiting component for the pheromone response and support the idea that beta and gamma subunits act as a positive regulator. Furthermore, overexpression of GPA1 prevented cell-cycle arrest but not FUS1 induction mediated by overexpression of STE4. This implies that the alpha subunit acts as a negative regulator presumably through interacting with beta and gamma subunits in the mating pheromone signaling pathway.     

G protein signaling governing cell fate decisions involves opposing Galpha subunits in Cryptococcus neoformans

Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Galpha subunits, most filamentous ascomycetes and basidiomycetes have three Galpha subunits. Previous studies have defined the Galpha subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Galpha subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Galpha subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Galpha into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development.     

Dominant negative mutations in the α-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product.     

Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.     

The yeast G-protein homolog is involved in the mating pheromone signal transduction system.

I have isolated a new type of sterile mutant of Saccharomyces cerevisiae, carrying a single mutant allele, designated dac1, which was mapped near the centromere on chromosome VIII. The dac1 mutation caused specific defects in the pheromone responsiveness of both a and alpha cells and did not seem to be associated with any pleiotropic phenotypes. Thus, in contrast to the ste4, ste5, ste7, ste11, and ste12 mutations, the dac1 mutation had no significant effect on such constitutive functions of haploid cells as pheromone production and alpha-factor destruction. The characteristics of this phenotype suggest that the DAC1 gene encodes a component of the pheromone response pathway common to both a and alpha cells. Introduction of the GPA1 gene encoding an S. cerevisiae homolog of the alpha subunit of mammalian guanine nucleotide-binding regulatory proteins (G proteins) into sterile dac1 mutants resulted in restoration of pheromone responsiveness and mating competence to both a and alpha cells. These results suggest that the dac1 mutation is an allele of the GPA1 gene and thus provide genetic evidence that the yeast G protein homolog is directly involved in the mating pheromone signal transduction pathway.     

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product. Received: 15 January 1999 / Accepted: 25 March 1999     

Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response.     

Biochemical analysis of yeast G(alpha) mutants that enhance adaptation to pheromone

The mating-specific heterotrimeric G(alpha) protein of Saccharomyces cerevisiae, Gpa1, negatively regulates activation of the pheromone response pathway both by sequestering G(beta)gamma and by triggering an adaptive response through an as yet unknown mechanism. Previous genetic studies identified mutant alleles of GPA1 that downregulate the pheromone response independently of the pheromone receptor (GPA1E364K), or through a receptor-dependent mechanism (GPA1N388D). To further our understanding of the mechanism of action of these mutant alleles, their corresponding proteins were purified and subjected to biochemical analysis. The receptor-dependent activity of Gpa1N388D was further analyzed using yeast strains expressing constitutively active receptor (Ste2) mutants, and C-terminal truncation mutant forms of Gpa1. A combination of G(alpha) affinity chromatography, GTP binding/hydrolysis studies, and genetic analysis allowed us to assign a distinct mechanism of action to each of these mutant proteins.     

Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response. Received: 4 May 1998 / Accepted: 24 July 1998     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.     

Dual lipid modification motifs in G(alpha) and G(gamma) subunits are required for full activity of the pheromone response pathway in Saccharomyces cerevisiae

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein gamma subunit (Ste18p) is unusual among G(gamma) subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G(gamma) subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G(betagamma) after receptor-stimulated release from G(alpha). The G protein alpha subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.     

Switch-domain mutations in the Saccharomycescerevisiae G protein α-subunit Gpa1p identify a receptor subtype-biased mating defect

The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (α subunit), Ste4p (β) and Ste18p (γ). The switch II region of Gα subunits is involved in several protein-protein interactions and an intrinsic GTPase activity. To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations. The Q323 analog in Gα subunits and Ras is implicated in GTP hydrolysis. Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity. Mutation of residue A59 of Ras and the analogous Gαs residue has had quite different effects. The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in α cells than in a cells. In addition the G321T mutation resulted in an unusual pheromone response phenotype. We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and α-factor receptors.     

G protein mutations that alter the pheromone response in Saccharomyces cerevisiae.

The GPA1 gene of Saccharomyces cerevisiae encodes a G alpha protein that couples the membrane-bound pheromone receptors to downstream elements in the mating response pathway. We have isolated seven mutant alleles of GPA1 that confer pheromone resistance: G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K, G470D, and an E364K-G470D double mutant. All of the mutations lie within large regions that are highly conserved between Gpa1 and four other G alpha proteins; four of the changes are located in domains with proposed functions. On the basis of a gentic analysis, the pheromone-unresponsive GPA1 alleles can be divided into two classes: those that encode constitutively activated proteins and those that encode proteins unable to respond to the upstream signal. Our results support the hypothesis that the activated form of Gpa1 stimulates adaptation to pheromone.     

Genetic identification of residues involved in association of alpha and beta G-protein subunits.

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpa1 protein. A mutation at position E307 of the Gpa1 protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant alpha subunit to physically associate with a specific mutant G beta subunit. The mutation in the Gpa1 protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the alpha subunit with the wild-type beta subunit. Yeast cells constructed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified G alpha subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.     

The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.     

GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway.

The yeast RAS1 and RAS2 genes appear to be involved in control of cell growth in response to nutrients. Here we show that this growth control also involves a signal mediated by the heterotrimeric G protein alpha subunit homolog encoded by GPA2. A GPA2 null allele conferred a severe growth defect on cells containing a null allele of RAS2, although either mutation alone had little effect on growth rate. A constitutive allele of GPA2 could stimulate growth of a strain lacking both RAS genes. Constitutive GPA2 conferred heat shock sensitivity on both wild-type cells and cells lacking RAS function, but had no effect in a strain containing a null allele of SCH9, which encodes a kinase related to protein kinase A. The GPR1 gene was isolated and was found to encode a protein with the characteristics of a G protein-coupled receptor. Double Deltagpr1 Deltaras2 mutants displayed a severe growth defect that was suppressed by expression of the constitutive allele of GPA2, confirming that GPR1 acts upstream of GPA2. Gpr1p is expressed on the cell surface and requires sequences in the membrane-proximal region of its third cytoplasmic loop for function, as expected for a G protein-coupled receptor. GPR1 RNA was induced when cells were starved for nitrogen and amino acids. These results are consistent with a model in which the GPR1/GPA2 pathway activates the Sch9p kinase to generate a response that acts in parallel with that generated by the Ras/cAMP pathway, resulting in the integration of nutrient signals.     

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.