Cyclic AMP-protein kinase A and Snf1 signaling mechanisms underlie the superior potency of sucrose for induction of filamentation in Saccharomyces cerevisiae

Under specific environmental conditions, the yeast Saccharomyces cerevisiae can undergo a morphological switch to a pseudohyphal growth pattern. Pseudohyphal differentiation is generally studied upon induction by nitrogen limitation in the presence of glucose. It is known to be controlled by several signaling pathways, including mitogen-activated protein kinase, cyclic AMP-protein kinase A (cAMP-PKA), and Snf1 kinase pathways. We show that the alpha-glucoside sugars maltose and maltotriose, and especially sucrose, are more potent inducers of filamentation than glucose. Sucrose even induces filamentation in nitrogen-rich media and in the mep2Δ/mep2Δ ammonium permease mutant on ammonium-limiting medium. We demonstrate that glucose also inhibits filamentation by means of a pathway parallel to the cAMP-PKA pathway. Deletion of HXK2 shifted the pseudohyphal growth pattern on glucose to that of sucrose, while deletion of SNF4 abrogated filamentation on both sugars, indicating a negative role of glucose repression and a positive role for Snf1 activity in the control of filamentation. In all strains and in all media, sucrose induction of filamentation is greatly diminished by deletion of the sucrose/glucose-sensing G-protein-coupled receptor Gpr1, whereas it has no effect on induction by maltose and maltotriose. The competence of alpha-glucoside sugars to induce filamentation is reflected in the increased expression of the cell surface flocculin gene FLO11. In addition, sucrose is the only alpha-glucoside sugar capable of rapidly inducing FLO11 expression in a Gpr1-dependent manner, reflecting the sensitivity of Gpr1 for this sugar and its involvement in rapid sucrose signaling. Our study identifies sucrose as the most potent nutrient inducer of pseudohyphal growth and shows that glucose inactivation of Snf1 kinase signaling is responsible for the lower potency of glucose.     

Snf1 kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth

The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different beta-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.     

Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains.

Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the Galpha subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of Deltamep2/Deltamep2 and Deltagpa2/Deltagpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in Deltamep1/Deltamep1 Deltamep2/Deltamep2 and Deltamep1/Deltamep1 Deltagpa2/Deltagpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a Deltamep1/Deltamep1 Deltamep2/Deltamep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth.     

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.     

Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway

Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 mitogen-activated protein (MAP) kinase cascade, which is composed of three tiers of protein kinases: (i) the SSK2, SSK22, and STE11 MAP kinase kinase kinases (MAPKKKs), (ii) the PBS2 MAPKK, and (iii) the HOG1 MAP kinase. Activation of the MAP kinase cascade is mediated by two upstream mechanisms. The SLN1-YPD1-SSK1 two-component osmosensor activates the SSK2 and SSK22 MAPKKKs by direct interaction of the SSK1 response regulator with these MAPKKKs. The second mechanism of HOG1 MAP kinase activation is independent of the two-component osmosensor and involves the SHO1 transmembrane protein and the STE11 MAPKKK. Only PBS2 and HOG1 are common to the two mechanisms. We conducted an exhaustive mutant screening to identify additional elements required for activation of STE11 by osmotic stress. We found that strains with mutations in the STE50 gene, in combination with ssk2Δ ssk22Δ mutations, were unable to induce HOG1 phosphorylation after osmotic stress. Both two-hybrid analyses and coprecipitation assays demonstrated that the N-terminal domain of STE50 binds strongly to the N-terminal domain of STE11. The binding of STE50 to STE11 is constitutive and is not affected by osmotic stress. Furthermore, the two proteins relocalize similarly after osmotic shock. It was concluded that STE50 fulfills an essential role in the activation of the high-osmolarity glycerol response pathway by acting as an integral subunit of the STE11 MAPKKK.     

A third osmosensing branch in Saccharomyces cerevisiae requires the Msb2 protein and functions in parallel with the Sho1 branch

Two Saccharomyces cerevisiae plasma membrane-spanning proteins, Sho1 and Sln1, function during increased osmolarity to activate a mitogen-activated protein (MAP) kinase cascade. One of these proteins, Sho1, utilizes the MAP kinase kinase kinase Ste11 to activate Pbs2. We previously used the FUS1 gene of the pheromone response pathway as a reporter to monitor cross talk in hog1 mutants. Cross talk requires the Sho1-Ste11 branch of the HOG pathway, but some residual signaling, which is STE11 dependent, still occurs in the absence of Sho1. These observations led us to propose the existence of another osmosensor upstream of Ste11. To identify such an osmosensor, we screened for mutants in which the residual signaling in a hog1 sho1 mutant was further reduced. We identified the MSB2 gene, which encodes a protein with a single membrane-spanning domain and a large presumptive extracellular domain. Assay of the FUS1-lacZ reporter (in a hog1 mutant background) showed that sho1 and msb2 mutations both reduced the expression of the reporter partially and that the hog1 sho1 msb2 mutant was severely defective in the expression of the reporter. The use of DNA microarrays to monitor gene expression revealed that Sho1 and Msb2 regulate identical gene sets in hog1 mutants. A role for MSB2 in HOG1 strains was also seen in strains defective in the two known branches that activate Pbs2: an ssk1 sho1 msb2 strain was more osmosensitive than an ssk1 sho1 MSB2 strain. These observations indicate that Msb2 is partially redundant with the Sho1 osmosensing branch for the activation of Ste11.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.     

Crosstalk between cAMP-PKA and MAP kinase pathways is a key regulatory design necessary to regulate FLO11 expression

Signal transduction pathways crosstalk with one another and play a central role in regulation of cellular events. Crosstalk brings complexity to the system, and hence, a systematic analysis of these crosstalks helps in relating the signaling network structure to its function. Here, we present a modular steady state approach to quantify the network comprising of cAMP-PKA and MAP kinase pathways involved in the regulation of FLO11, a gene which is required for pseudohyphae growth in Saccharomyces cerevisiae under nitrogen starvation. These two pathways crosstalk by converging on the same target, i.e., FLO11 and through Ras2p, an upstream activator of both cAMP and MAPK pathway. Analysis of crosstalk at the gene level revealed that cAMP-PKA and MAPK pathways are indispensable to FLO11 expression. The dose response was highly sensitive and primarily controlled by cAMP-PKA pathway. We demonstrate that the highly sensitive response in the cAMP-PKA pathway was due to crosstalk and inhibitor ultrsensitivity, key regulatory designs present at the downstream of cAMP-PKA pathway. The analysis of the role of Ras2p in the crosstalk between the cAMP-PKA and MAPK pathways indicated that crosstalk essentially helped in amplification of the Gpa2p signal, another upstream activator of the cAMP-PKA pathway. However, the effect of crosstalk due to Ras2p on FLO11 expression was minimal under normal activation levels of Ras2p. Whereas, the crosstalk itself can bring about FLO11 expression under the hyperactivated Ras2p conditions thereby eliminating the requirement for the other activator Gpa2p. We also observed the presence of system level properties such as amplification, inhibitor ultrasensitvity and bistability, which can be attributed to the regulatory design present in the FLO11 expression system. These system level properties might help the organism to respond to varying nutritional status.     

Identification of SFL1 as a positive regulator for flor formation in Zygosaccharomyces rouxii

ABSTRACT

Some wild Zygosaccharomyces rouxii impair the quality of soy sauce through the generation of unpleasant odors induced by the formation of flor. Flor formation in Z. rouxii depends on the expression of the FLO11D gene, which is a homolog of the FLO11 gene that encodes a cell surface protein in Saccharomyces cerevisiae. FLO11 expression in S. cerevisiae is regulated by multiple pathways. To investigate the regulation of FLO11D expression in Z. rouxii, we created 13 gene knockout mutants (STE12, TEC1, HOG1, MSS11, FLO8, MSN1, MSN2/4, SKO1, TUP1, CYC8, YAK1, MIG1, and SFL1) related to those pathways and examined whether these mutants form flor. Unexpectedly, SFL1 knockout mutant could only form a very weak flor due to decreased FLO11D expression, suggesting that SFL1 acts as a potential activator of flor formation through FLO11D expression. This result is in contrast to S. cerevisiae SFL1, which acts as a repressor of FLO11 expression.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.Communicated by C. P. Hollenberg