Analysis of RGS Proteins in Saccharomyces cerevisiae

Hoffman, Ginger A.; Garrison, Tiffany Runyan; Dohlman, Henrik G.

doi:10.1016/s0076-6879(02)44744-1

Cited by 79 publications

(89 citation statements)

References 9 publications

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“…Alterations in pheromone sensitivity were measured using a reporter transcription assay comprised of a pheromone-responsive promoter (from FUS1) fused to lacZ (␤-galactosidase). This particular assay was chosen because it is sensitive, quantitative, and highly specific for Gpa1 signaling (22). We chose not to use fluorescence-based measures of G protein dissociation because they require modifications or fusions that might alter G protein stability, localization, or activity (29).…”

Section: Resultsmentioning

confidence: 99%

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Regulators of G Protein Signaling and Transient Activation of Signaling

Hao

Yıldırım

Wang

et al. 2003

Journal of Biological Chemistry

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Cellular responses to hormones and neurotransmitters are necessarily transient. The mating pheromone signal in yeast is typical. Signal initiation requires cell surface receptors, a G protein heterotrimer, and downstream effectors. Signal inactivation requires Sst2, a regulator of G protein signaling (RGS) protein that accelerates GTPase activity. We conducted a quantitative analysis of RGS and G protein expression and devised computational models that describe their activity in vivo. These results indicated that pheromone-dependent transcriptional induction of the RGS protein constitutes a negative feedback loop that leads to desensitization. Modeling also suggested the presence of a positive feedback loop leading to resensitization of the pathway. In confirmation of the model, we found that the RGS protein is ubiquitinated and degraded in response to pheromone stimulation. We identified and quantitated these positive and negative feedback loops, which account for the transient response to external signals observed in vivo.One measure of our understanding of biological systems is our ability to predict their behavior in detail. One aspect of this endeavor is to model signal transduction events, defined here as the dynamic changes that occur within a cell in response to an external stimulus (1-3). Such models can help us to understand how small changes outside a cell produce strongly amplified changes within a cell, how graded signals are converted to all-or-none responses (4), or how activators of one pathway influence the function of a second pathway (5). A second goal, and the focus of this work, is to understand how transient external signals are prevented from being propagated indefinitely within the cell. Here we describe the molecular basis for signal activation, desensitization, and eventual resensitization of G proteins by receptors and RGS 1 proteins. The experimentally observed behavior is described mechanistically by computational modeling of the pathway.For these studies we investigated the mating pheromone signaling pathway in yeast Saccharomyces cerevisiae. The yeast mating response is arguably the best characterized signal transduction pathway of any eukaryote, and it has long served as a prototype for hormone, neurotransmitter, and sensory response systems in humans (6). Disruption or activation of pathway components leads to highly specific changes that can be easily quantified. Finally, because it is a unicellular eukaryote, every cell in a population is genetically and phenotypically identical (all cells are "typical").Mating in yeast is the fusion of a and ␣ haploid cell types to form an a/␣ diploid. The events leading to fusion are initiated by specific pheromones: ␣-type cells secrete ␣-factor pheromone, which binds to a specific receptor (Ste2) on a-cells, while a-cells secrete a-factor that binds to receptors (Ste3) on ␣-cells. Upon pheromone binding to its receptor, the G protein ␣ subunit (Gpa1) releases GDP, binds to GTP, and liberates the G protein ␤␥ subunits (Ste4/Ste18). Sustained...

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Section: Resultsmentioning

confidence: 99%

“…Signaling, Expression, Ubiquitination, and Degradation AssaysThe pheromone-dependent transcription assays using the ␤-galactosidase (22) and green fluorescent protein (23) reporters have been described previously. For immunoblot detection, cell growth was stopped by the addition of 10 mM NaN 3 and transfer to an ice bath.…”

Section: Methodsmentioning

confidence: 99%

Regulators of G Protein Signaling and Transient Activation of Signaling

Hao

Yıldırım

Wang

et al. 2003

Journal of Biological Chemistry

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“…Parental and allele-replacement strains were transformed with the plasmid pGA1706 (56) containing three copies of the PRE, which drives expression of the lacZ reporter. Measurement of fluorescein was carried out as previously described (57). When the DIG2 allele from RM is substituted in BY, the degree of induction is even stronger.…”

Section: Methodsmentioning

confidence: 99%

Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets

Fazlollahi

Muroff

Lee

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Regulation of gene expression by transcription factors (TFs) is highly dependent on genetic background and interactions with cofactors. Identifying specific context factors is a major challenge that requires new approaches. Here we show that exploiting natural variation is a potent strategy for probing functional interactions within gene regulatory networks. We developed an algorithm to identify genetic polymorphisms that modulate the regulatory connectivity between specific transcription factors and their target genes in vivo. As a proof of principle, we mapped connectivity quantitative trait loci (cQTLs) using parallel genotype and gene expression data for segregants from a cross between two strains of the yeast Saccharomyces cerevisiae. We identified a nonsynonymous mutation in the DIG2 gene as a cQTL for the transcription factor Ste12p and confirmed this prediction empirically. We also identified three polymorphisms in TAF13 as putative modulators of regulation by Gcn4p. Our method has potential for revealing how genetic differences among individuals influence gene regulatory networks in any organism for which gene expression and genotype data are available along with information on binding preferences for transcription factors.

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“…Pheromone sensitivity halo assays were performed as described elsewhere (Hoffman et al, 2002), using the strains of interest on selective inducing plates. The Dfus3 strain was used as a control.…”

Section: Methodsmentioning

confidence: 99%

“…6b). We then performed a halo assay to determine whether the effect of HopX1 on the pheromone MAPK pathway has an impact on yeast sensitivity to afactor (Hoffman et al, 2002). Cells expressing HopX1 or containing an empty vector were able to produce haloes of similar size in response to a-factor, as opposed to cells in which Fus3, the MAPK of the pheromone-regulated pathway, was deleted (Fig.…”

Section: Effects Of Hopx1 On the Activation Of Other Mapk Pathways Inmentioning

confidence: 99%

Expression of Pseudomonas syringae type III effectors in yeast under stress conditions reveals that HopX1 attenuates activation of the high osmolarity glycerol MAP kinase pathway

et al. 2012

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The Gram-negative bacterium Pseudomonas syringae pv. tomato (Pst) is the causal agent of speck disease in tomato. Pst pathogenicity depends on a type III secretion system that delivers effector proteins into host cells, where they promote disease by manipulating processes to the advantage of the pathogen. Previous studies identified seven Pst effectors that inhibit growth when expressed in yeast under normal growth conditions, suggesting that they interfere with cellular processes conserved in yeast and plants. We hypothesized that effectors also target conserved cellular processes that are required for yeast growth only under stress conditions. We therefore examined phenotypes induced by expression of Pst effectors in yeast grown in the presence of various stressors. Out of 29 effectors tested, five (HopX1, HopG1, HopT1-1, HopH1 and AvrPtoB) displayed growth inhibition phenotypes only in combination with stress conditions. Viability assays revealed that the HopX1 effector caused loss of cell viability under prolonged osmotic stress. Using transcription reporters, we found that HopX1 attenuated the activation of the high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway, which is responsible for yeast survival under osmotic stress, while other MAPK pathways were mildly affected by HopX1. Interestingly, HopX1-mediated phenotypes in yeast were dependent on the putative transglutaminase catalytic triad of the effector. This study enlarges the pool of phenotypes available for the functional analysis of Pst type III effectors in yeast, and exemplifies how analysis of phenotypes detected in yeast under stress conditions can lead to the identification of eukaryotic cellular processes affected by bacterial effectors.

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Analysis of RGS Proteins in Saccharomyces cerevisiae

Cited by 79 publications

References 9 publications

Regulators of G Protein Signaling and Transient Activation of Signaling

Regulators of G Protein Signaling and Transient Activation of Signaling

Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets

Expression of Pseudomonas syringae type III effectors in yeast under stress conditions reveals that HopX1 attenuates activation of the high osmolarity glycerol MAP kinase pathway

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