Constitutive mutants in the yeast pheromone response: Ordered function of the gene products

Blinder, Dmitry B.; Bouvier, Suzanne E.; Jenness, Duane D.

doi:10.1016/0092-8674(89)90250-x

Cited by 137 publications

(95 citation statements)

References 38 publications

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“…Thus, the primary role for Ste5 as a nucleation site for the assembly of protein kinases involved in signal transduction is consistent with the previous epistasis analyses (MacKay 1983;Blinder et al 1989;Elion et al 1991a, b;Cairns et al 1992;Leberer et al 1992Leberer et al , 1993Stevenson et al 1992;Hasson et al 1994;Ramer and Davis 1993), if we assume that the enzymatic reactions, but not necessarily the physical associations, are in a linear series. The fact that Ste5 is so limiting for Fus3 activation may suggest Ste5 is in low abundance, consistent with a critical role as a potent activator.…”

Section: Evidence Of a Direct Role For Stes In Fuss Activation: A Modsupporting

confidence: 88%

“…Based on genetic data, these signal transduction components have been ordered into a linear pathway upstream of the transcription factor, Stell, that is activated and phosphorylated in response to pheromone (Dolan et al 1989;Dolan and Fields 1990). In this linear pathway model, signal transduction initiates by pheromone binding to a cell surface receptor, activating a heterotrimeric G-protein, then continues successively through the putative protein kinase Ste20, the SteS protein, the protein kinases Stell and Ste7, and culminating in the activation of Fus3 and Kssl, related protein kinases that activate Stel2 (Blinder et al 1989;Elion et al 1990Elion et al , 1991bWhiteway et al 1990;Cairns et al 1992;Gartner et al ^Present address: Cubist Phannaceuticals, 24 Emily Street, Cambridge, MA 02139 USA. ^Conesponding author.…”

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confidence: 99%

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The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5.

Kranz¹,

Satterberg²,

Elion³

1994

Genes Dev.

124

107

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Activation of the Saccbaromyces cerevisiae MAP kinase Fus3 is thought to occur via a linear pathway involving the sequential action of three proteins: Ste5, a protein of unknown function, Stell, a MAPKK kinase homology and Ste7, a MAPK kinase homolog which phosphorylates and activates Fus3. In this report, we present evidence for a novel mechanism of Fus3 activation that involves a direct association with Ste5, a protein not predicted to interact with Fus3. First, overexpression of Ste5 suppresses fus3 point mutations in an allele-specific manner and increases Fus3 kinase activity in vitro. Second, Ste5 associates with Fus3 in vivo as demonstrated by the two-hybrid system and by two methods of copurification. Third, Ste5 and Fus3 associate prior to pheromone stimulation even when Fus3 is inactive, and in strains lacking Ste7 and Stell. Fourth, Ste5 is phosphorylated by Fus3 in purified complexes and copurifies with an additional protein kinase(s). These observations suggest the possibility that SteS promotes signal transduction by tethering Fus3 to its activating protein kinase(s).

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Section: Evidence Of a Direct Role For Stes In Fuss Activation: A Modsupporting

confidence: 88%

mentioning

confidence: 99%

The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5.

Kranz¹,

Satterberg²,

Elion³

1994

Genes Dev.

124

107

View full text Add to dashboard Cite

show abstract

“…This organism has several well-known advantages for investigating cellular phenomena, including those associated with the plasma membrane (Botstein & Fink, 1988). Molecular events of S. cerevisiae transmembrane signalling by mating pheromones are particularly relevant (Blinder et al, 1989;Herskowitz & Marsh, 1987;Jahng et al, 1988;Nakayama et al, 1988). These events may be deduced by isolating and observing the phenotypes of mutants altered in the response to pheromones and identifying the impaired genes.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of action of the phytotoxin syringomycin: a resistant mutant of Saccharomyces cerevisiae reveals an involvement of Ca2+ transport

Takemoto

Zhang

Taguchi

et al. 1991

Journal of General Microbiology

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The bacterial phytotoxin syringomycin affects plasma-membrane-associated functions of plants and yeast. These include increases in transmembrane K+, H+ and Ca2+ fluxes and membrane potential. Mutants of Saccharomyces cerevisiae resistant to growth inhibition by syringomycin were isolated and characterized. Many of the mutant isolates were unable to grow in yeast extract/peptone/dextrose medium supplemented with 400 mM-CaC1, which permitted the growth of the parent strain (8A-1B). Genetic analyses of one of these mutants, strain R4-3G, showed a single recessive mutation that simultaneously led to Ca2+-sensitivity and syringomycin-resistance. R4-3G had higher net 45Ca2+ uptake rates than strain 8A-1B and higher intracellular Ca2+ levels in medium containing 1 mMCaCl,. The,aJtered 45Ca2+ uptake rates of the mutant were not influenced by syringomycin and were not related to altered capabilities for Ca2+ efflux. R4-3G had similar syringomycin-stimulated increases in K+ efflux but lower syringomycin-stimulated increases in membrane potential than 8A-1B. It is concluded that Ca2+ transport is important in the response of yeast to syringomycin and that the toxin-stimulated membrane potential increase, but not K+ efflux, is closely associated with Ca2+ transport.

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“…Operationally, this objective could be accomplished by expressing the foreign gene in yeast from a yeast plasmid, mutagenizing the yeast strain, and then screening for a plasmid-dependent yeast mutant. [Screening for plasmid dependence has been quite successful in other contexts in yeast (8)(9)(10).] Cloning the yeast gene by using this yeast mutant would then be straightforward with standard techniques (11).…”

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confidence: 99%

Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.

Kranz

Holm

1990

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

141

114

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Studies of cell physiology and structure have identified many intriguing proteins that could be analyzed for function by using the power of yeast genetics. Unfortunately, identifying the homologous yeast gene with the two most commonly used approaches-DNA hybridization and antibody cross-reaction-is often difficult. We describe a strategy to identify yeast homologs based on protein function itself. This cloning-by-function strategy involves first identifying a yeast mutant that depends on a plasmid expressing a cloned foreign gene. The corresponding yeast gene is then cloned by complementation of the mutant defect. To detect plasmid dependence, the colony color assay of Koshland et al. [Koshland, D., Kent, J. C. & Hartwell, L. H. (1985) Cell 40,[393][394][395][396][397][398][399][400][401][402][403] is used. In this paper, we test the feasibility of this approach using the wellcharacterized system of DNA topoisomerase II in yeast. We show that (i) plasmid dependence is easily recognized; (ii) the screen efficiently isolates mutations in the desired gene; and (iii) the wild-type yeast homolog of the gene can be cloned by screening for reversal of the plasmid-dependent phenotype. We conclude that cloning by function can be used to isolate the yeast homologs of essential genes identified in other organisms.Many genes have been isolated that encode products essential for eukaryotic cells. The difficulty of genetic analysis in higher eukaryotes makes it attractive to study such genes by using the extraordinary power of yeast genetics. Unfortunately, because of the evolutionary distance between yeast and other experimental organisms, it is often difficult to identify the yeast homolog of a protein from another species by using the standard approaches of DNA hybridization and cross-species antibody reaction.Recent successes with substituting genes from other organisms for yeast genes suggest that an alternative approach will be very productive. Many examples are now available of foreign cDNAs encoding products that substitute fully for yeast proteins inactivated by mutation. For example, the human homolog of a Schizosaccharomyces pombe mitotic control gene, cdc2, was cloned by introducing a human cDNA library into a cdc2'5 strain and then screening for growth at the restrictive temperature (1). Additional examples of such conservation of function through evolution include other cell cycle proteins (2), protein transport components (3, 4), cytochrome c (5), and transcription factors (6, 7). The properties of the proteins in these examples suggest that not only monomer function, but also protein-protein interactions, have been conserved in evolution. The case of cytochrome c is particularly striking, not only because of the complex processing pathway that must be followed to produce a completely functional protein, but also because antisera raised against yeast or mouse cytochrome c do not react with the alternative cytochrome c; furthermore, DNA probes from yeast or rat cytochrome c genes do not cross-hybridize ...

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Constitutive mutants in the yeast pheromone response: Ordered function of the gene products

Cited by 137 publications

References 38 publications

The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5.

The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5.

Mechanism of action of the phytotoxin syringomycin: a resistant mutant of Saccharomyces cerevisiae reveals an involvement of Ca2+ transport

Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.

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