Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.     

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.     

Unstructured N terminus of the RNA polymerase II subunit Rpb4 contributes to the interaction of Rpb4.Rpb7 subcomplex with the core RNA polymerase II of Saccharomyces cerevisiae

Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4.Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.     

The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth.

Saccharomyces cerevisiae mutant E124 was selected in a visual screen based on elongated cell shape. Genetic analysis showed that E124 contains two separate mutations, pps1-1 and elm4-1, each causing a distinct phenotype inherited as a single-gene trait. In rich medium, pps1-1 by itself causes increased doubling time but does not affect cell shape, whereas elm4-1 results in a moderate cell elongation phenotype but does not affect growth rate. Reconstructed elm4-1 pps1-1 double mutants display a synthetic phenotype in rich medium including extreme cell elongation and delayed cell separation, both characteristics of pseudohyphal differentiation. The elm4-1 mutation was shown to act as a dominant factor that potentiates pseudohyphal differentiation in response to general nitrogen starvation in a genetic background in which pseudohyphal growth normally does not occur. Thus, elm4-1 allows recognition of, or response to, a pseudohyphal differentiation signal that results from nitrogen limitation. PPS1 was isolated and shown to be a previously undescribed gene coding for a protein similar in amino acid sequence to phosphoribosylpyrophosphate synthase, a rate-limiting enzyme in the biosynthesis of nucleotides, histidine, and tryptophan. Thus, the pps1-1 mutation may generate a nitrogen limitation signal, which when coupled with elm4-1 results in pseudohyphal growth even in rich medium.     

Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p. Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control.     

Mapping the interaction site of Rpb4 and Rpb7 subunits of RNA polymerase II in Saccharomyces cerevisiae

Rpb4 and Rpb7, the fourth and the seventh largest subunits of RNA polymerase II, form a heterodimer in Saccharomyces cerevisiae. To identify the site of interaction between these subunits, we constructed truncation mutants of both these proteins and carried out yeast two hybrid analysis. Deletions in the amino and carboxyl terminal domains of Rpb7 abolished its interaction with Rpb4. In comparison, deletion of up to 49 N-terminal amino acids of Rpb4 reduced its interaction with Rpb7. Complete abolishment of interaction between Rpb4 and Rpb7 occurred by truncation of 1-106, 1-142, 108-221, 172-221 or 198-221 amino acids of Rpb4. Use of the yeast two-hybrid analysis in conjunction with computational analysis of the recently reported crystal structure of Rpb4/Rpb7 sub-complex allowed us to identify regions previously not suspected to be involved in the functional interaction of these proteins. Taken together, our results have identified the regions that are involved in interaction between the Rpb4 and Rpb7 subunits of S. cerevisiae RNA polymerase II in vivo.     

A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae

Methyl-benzimidazole-2-ylcarbamate (MBC) inhibits the mitotic cell cycle of Saccharomyces cerevisiae at a stage subsequent to DNA synthesis and before the completion of nuclear division (Quinlan, R. A., C. I. Pogson, and K, Gull, 1980, J Cell Sci., 46: 341-352). The step in the cell cycle that is sensitive to MBC inhibition was ordered to reciprocal shift experiments with respect to the step catalyzed by cdc gene products. Execution of the CDC7 step is required for the initiation of DNA synthesis and for completion of the MBC-sensitive step. Results obtained with mutants (cdc2, 6, 8, 9, and 21) defective in DNA replication and with an inhibitor of DNA replication (hydroxyurea) suggest that some DNA replication required for execution of the MBC-sensitive step but that the completion of replication is not. Of particular interest were mutants (cdc5, 13, 14, 15, 16, 17, and 23) that arrest cell division after DNA replication but before nuclear division since previous experiments had not been able to resolve the pathway of events in this part of the cell cycle. Execution of the CDC17 step was found to be a prerequisite for execution of the MBC- sensitive step; the CDC13, 16 and 23 steps are executed independently of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the CDC14 and 23 steps. These results considerably extend the dependent pathway of events that constitute the cell cycle of S. cerevisiae.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

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.     

Regulation of mat responses by a differentiation MAPK pathway in Saccharomyces cerevisiae

Fungal species exhibit diverse behaviors when presented with extracellular challenges. Pathogenic fungi can undergo cell differentiation and biofilm formation in response to fluctuating nutrient levels, and these responses are required for virulence. In the model fungal eukaryote Saccharomyces cerevisiae, nutrient limitation induces filamentous growth and biofilm/mat formation. Both responses require the same signal transduction (MAPK) pathway and the same cell adhesion molecule (Flo11) but have been studied under different conditions. We found that filamentous growth and mat formation are aspects of a related response that is regulated by the MAPK pathway. Cells in yeast-form mats differentiated into pseudohyphae in response to nutrient limitation. The MAPK pathway regulated mat expansion (in the plane of the XY-axis) and substrate invasion (downward in the plane of the Z-axis), which optimized the mat's response to extracellular nutrient levels. The MAPK pathway also regulated an upward growth pattern (in the plane of the Z-axis) in response to nutrient limitation and changes in surface rigidity. Upward growth allowed for another level of mat responsiveness and resembled a type of colonial chemorepulsion. Together our results show that signaling pathways play critical roles in regulating social behaviors in which fungal cells participate. Signaling pathways may regulate similar processes in pathogens, whose highly nuanced responses are required for virulence.     

Endogenous xylose pathway in Saccharomyces cerevisiae

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source.