Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

Dohlman, Henrik G.; Goldsmith, Paul K.; Spiegel, Allen M.; Thorner, Jeremy

doi:10.1073/pnas.90.20.9688

Cited by 54 publications

(49 citation statements)

References 64 publications

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“…Calculated expression levels of endogenous Gpa1 and Sst2 with (ϩ) and without (Ϫ) 1-h treatment with 3 M ␣-factor are summarized in Table I. Note that a portion of endogenous Gpa1 and none of the Escherichia coli-expressed Gpa1 is myristoylated (Myr-Gpa1) as reported previously (24,44). B, whole-cell extracts were prepared from wild-type (BY4741) cells transformed with a single copy plasmid (pRS316) containing genomic SST2 (2xSST2) or no insert and treated with 3 M ␣-factor for the indicated times.…”

Section: Resultsmentioning

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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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%

Regulators of G Protein Signaling and Transient Activation of Signaling

Hao

Yıldırım

Wang

et al. 2003

Journal of Biological Chemistry

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“…Hence, the GPA1 moiety on the fusions was able to compensate for overexpression of STE4. Myristoylation of Gpa1 is necessary for repression of the pheromone pathway (13,19), and is believed to be required for association of G ␣ and G ␤␥ (20). The STE4-GPA1 fusion constructs clearly cannot be myristoylated, but the covalent association of the Ste4 and Gpa1 subunits apparently overrides the need for myristoylation.…”

Section: Resultsmentioning

confidence: 99%

Signal transduction by a nondissociable heterotrimeric yeast G protein

Klein¹

2000

Proceedings of the National Academy of Sciences

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Many signal transduction pathways involve heterotrimeric G proteins. The accepted model for activation of heterotrimeric G proteins states that the protein dissociates to the free G␣ (GTP)-bound subunit and free G␤␥ dimer. On GTP hydrolysis, G␣ (GDP) then reassociates with G␤␥ [Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649]. We reexamined this hypothesis, by using the mating G protein of the yeast Saccharomyces cerevisiae encoded by the genes GPA1, STE4, and STE18. In the absence of mating pheromone, the G␣ (Gpa1) subunit represses the mating pathway. On activation by binding of pheromone to a serpentine receptor, the G␤␥ (Ste4, Ste18) dimer transmits the signal to a mitogen-activated protein kinase cascade, leading to gene activation, arrest in the G1 stage of the cell cycle, production of shmoos (mating projections), and cell fusion. We found that a Ste4-Gpa1 fusion protein transmitted the pheromone signal and activated the mating pathway as effectively as when Ste4 (G␤) and Gpa1 (G␣) were coexpressed as separate proteins. Hence, dissociation of this G protein is not required for its activation. Rather, a conformational change in the heterotrimeric complex is likely to be involved in signal transduction.

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“…D. Jenness and J. Hirschman (University of Massachusetts Medical Center, Worcester, MA). Anti-G ␣ -subunit antibodies were as described (20). Anti-Pma1p antibodies were kindly provided by Dr. N. Nelson and anti-Gas1p antibodies by Dr. H. Riezman.…”

Section: Methodsmentioning

confidence: 99%

Identification of Triton X-100 Insoluble Membrane Domains in the Yeast Saccharomyces cerevisiae

Kübler

Dohlman

Lisanti

1996

Journal of Biological Chemistry

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Low density Triton X-100 insoluble (LDTI) membrane domains are found in most mammalian cell types. Previous biochemical and immunolocalization studies have revealed the presence of G-protein coupled receptors and heterotrimeric G-protein subunits (G ␣ and G ␤␥ subunits) within these structures, implicating mammalian LDTI membrane domains in G-protein coupled signaling. Here, we present biochemical evidence that similar LDTI structures exist in a genetically tractable organism, the yeast Saccharomyces cerevisiae. Yeast LDTI membranes were purified based on the known biochemical properties of mammalian LDTI membranes: (i) their Triton X-100 insolubility; and (ii) their discrete buoyant density in sucrose gradients. As with purified mammalian LDTI membranes, these yeast LDTI membranes harbor the subunits of the heterotrimeric G-proteins (G ␣ and G ␤␥ subunits). Other plasma membrane marker proteins (the plasma membrane H ؉ -ATPase and a GPIlinked protein Gas1p) are preferentially excluded from these purified fractions. Mutational and genetic analyses were performed to define the requirements for the targeting of G-protein subunits to these yeast membrane domains. We find that the targeting of G ␣ is independent of myristoylation, whereas targeting of G ␥ requires prenylation. Perhaps surprisingly, the targeting of G ␤ to this membrane domain did not require coexpression of G ␥ . It should now be possible to dissect the function of LDTI membrane domains using yeast as a model genetic system.

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Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae.

Cited by 54 publications

References 64 publications

Regulators of G Protein Signaling and Transient Activation of Signaling

Regulators of G Protein Signaling and Transient Activation of Signaling

Signal transduction by a nondissociable heterotrimeric yeast G protein

Identification of Triton X-100 Insoluble Membrane Domains in the Yeast Saccharomyces cerevisiae

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