Functional domains of the yeast STE12 protein, a pheromone-responsive transcriptional activator

1993 ◽  
Vol 13 (6) ◽  
pp. 3765-3772
Author(s):  
C Kirkman-Correia ◽  
I L Stroke ◽  
S Fields
Keyword(s):  
Transcriptional Activity ◽  
Protein A ◽  
Pheromone Response ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Proteins ◽  
Pheromone Response Pathway ◽  
Cell Type Specific ◽  
Carboxy Terminal ◽  
Induced Signal ◽  
Insertion Mutants

The pheromone response pathway of the yeast Saccharomyces cerevisiae is necessary for the basal level of transcription of cell-type-specific genes, as well as the induced level observed after pheromone treatment. The STE12 protein binds to the DNA sequence designated the pheromone response element and is a target of the pheromone-induced signal. We generated 6-nucleotide linker insertion mutants, internal-deletion mutants, and carboxy-terminal truncation mutants of STE12 and assayed them for their ability to restore mating and transcriptional activity to a ste12 delta strain. Two of these mutant proteins retain the capacity to mediate basal transcription but show little or no induced transcription upon pheromone treatment. Cells producing these proteins cannot mate, formally demonstrating that the ability to respond to pheromone by increasing gene expression is essential for the mating process. Since distinct domains of STE12 appear to be required for basal versus induced transcription, we suggest that the pheromone-induced signal is likely to target residues of the protein different from those targeted by the basal signal because of the constitutive activity of the response pathway. Our analysis of mutant STE12 proteins also indicates that only the DNA-binding domain is sensitive to the small changes caused by the linker insertions. In addition, we show that, while the carboxy-terminal sequences necessary for STE12 to form a complex with the transcription factor MCM1 are not essential for mating, these sequences are required for optimal transcriptional activity.

scholarly journals Functional domains of the yeast STE12 protein, a pheromone-responsive transcriptional activator.

1993 ◽  
Vol 13 (6) ◽  
pp. 3765-3772 ◽  
Author(s):  
C Kirkman-Correia ◽  
I L Stroke ◽  
S Fields
Keyword(s):  
Transcriptional Activity ◽  
Protein A ◽  
Pheromone Response ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Proteins ◽  
Pheromone Response Pathway ◽  
Cell Type Specific ◽  
Carboxy Terminal ◽  
Induced Signal ◽  
Insertion Mutants

The pheromone response pathway of the yeast Saccharomyces cerevisiae is necessary for the basal level of transcription of cell-type-specific genes, as well as the induced level observed after pheromone treatment. The STE12 protein binds to the DNA sequence designated the pheromone response element and is a target of the pheromone-induced signal. We generated 6-nucleotide linker insertion mutants, internal-deletion mutants, and carboxy-terminal truncation mutants of STE12 and assayed them for their ability to restore mating and transcriptional activity to a ste12 delta strain. Two of these mutant proteins retain the capacity to mediate basal transcription but show little or no induced transcription upon pheromone treatment. Cells producing these proteins cannot mate, formally demonstrating that the ability to respond to pheromone by increasing gene expression is essential for the mating process. Since distinct domains of STE12 appear to be required for basal versus induced transcription, we suggest that the pheromone-induced signal is likely to target residues of the protein different from those targeted by the basal signal because of the constitutive activity of the response pathway. Our analysis of mutant STE12 proteins also indicates that only the DNA-binding domain is sensitive to the small changes caused by the linker insertions. In addition, we show that, while the carboxy-terminal sequences necessary for STE12 to form a complex with the transcription factor MCM1 are not essential for mating, these sequences are required for optimal transcriptional activity.

Mutagenesis of Ste18, a putative Gγ subunit in the Saccharomyces cerevisiae pheromone response pathway

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1230-1237 ◽  
Author(s):  
Malcolm Whiteway ◽  
Daniel Dignard ◽  
David Y. Thomas
Keyword(s):  
Signal Transduction ◽  
Dominant Negative ◽  
Saturation Mutagenesis ◽  
Pheromone Response ◽  
Mutant Proteins ◽  
Pheromone Response Pathway ◽  
Allele Specific ◽  
Dominant Negative Mutations ◽  
Compensatory Mutations ◽  
Mutant Genes

The yeast STE18 gene product has sequence and functional similarity to the γ subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (Gβ) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the Gβ and Gγ subunits or in the association of the Gβγ subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPAI (Gα) gene. These mutant proteins may sequester Gβ or some other component of the signalling machinery in a nonfunctional complex. Key wordsi yeast, G protein, STE18, mutagenesis, pheromone response.

scholarly journals The carboxy-terminal tail of the Ste2 receptor is involved in activation of the G protein in the Saccharomyces cerevisiaeα-pheromone response pathway

2001 ◽  
Vol 197 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Ma.de Jesús Durán-Avelar ◽  
Laura Ongay-Larios ◽  
Alejandro Zentella-Dehesa ◽  
Roberto Coria
Keyword(s):  
G Protein ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Carboxy Terminal

scholarly journals Signal-mediated localization of Candida albicans pheromone response pathway components

2020 ◽  
Author(s):  
Anna Carolina Borges Pereira Costa ◽  
Raha Parvizi Omran ◽  
Chris Law ◽  
Vanessa Dumeaux ◽  
Malcolm Whiteway
Keyword(s):  
Candida Albicans ◽  
Map Kinase ◽  
Nuclear Localization ◽  
Map Kinases ◽  
Critical Role ◽  
Cell Periphery ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Localization Signal ◽  
In Cells

Abstract Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or β subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src

1990 ◽  
Vol 10 (4) ◽  
pp. 1307-1318
Author(s):  
H Hirai ◽  
H E Varmus
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Kinase Domain ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Positive Effects ◽  
Src Gene ◽  
Carboxy Terminal

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

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