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.     

Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling

Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth state with very few genetic and regulatory changes.     

Control of Saccharomyces cerevisiae Filamentous Growth by Cyclin-Dependent Kinase Cdc28

The ascomycete Saccharomyces cerevisiae exhibits alternative vegetative growth states referred to as the yeast form and the filamentous form, and it switches between the two morphologies depending on specific environmental signals. To identify molecules involved in control of morphologic differentiation, this study characterized mutant S. cerevisiae strains that exhibit filamentous growth in the absence of the normal external signals. A specific amino acid substitution in the cyclin-dependent protein kinase Cdc28 was found to cause constitutive expression of most filamentous growth characteristics. These effects include specifically modified cell polarity characteristics in addition to the defined shape and division cycle alterations typical of the filamentous form. Several other mutations affecting Cdc28 function also had specific effects on filamentous growth. Constitutive filamentous growth resulting from deletion of the protein kinase Elm1 was prevented by modification of Cdc28 such that it could not be phosphorylated on tyrosine residue 19. In addition, various mutations affecting Hsl1 or Swe1, known or presumed components of a protein kinase cascade that mediates Cdc28 phosphorylation on Y19, either prevented or enhanced filamentous growth. The data suggest that a protein kinase cascade involving Elm1, Hsl1, and Swe1 can modulate Cdc28 activity and that Cdc28 in turn exerts global effects that cause filamentous growth.     

Multiple TORC1-associated proteins regulate nitrogen starvation-dependent cellular differentiation in Saccharomyces cerevisiae

Background

The budding yeast Saccharomyces cerevisiae undergoes differentiation into filamentous-like forms and invades the growth medium as a foraging response to nutrient and environmental stresses. These developmental responses are under the downstream control of effectors regulated by the cAMP/PKA and MAPK pathways. However, the upstream sensors and signals that induce filamentous growth through these signaling pathways are not fully understood. Herein, through a biochemical purification of the yeast TORC1 (Target of Rapamycin Complex 1), we identify several proteins implicated in yeast filamentous growth that directly associate with the TORC1 and investigate their roles in nitrogen starvation-dependent or independent differentiation in yeast.

Methodology

We isolated the endogenous TORC1 by purifying tagged, endogenous Kog1p, and identified associated proteins by mass spectrometry. We established invasive and pseudohyphal growth conditions in two S. cerevisiae genetic backgrounds (Σ1278b and CEN.PK). Using wild type and mutant strains from these genetic backgrounds, we investigated the roles of TORC1 and associated proteins in nitrogen starvation-dependent diploid pseudohyphal growth as well as nitrogen starvation-independent haploid invasive growth.

Conclusions

We show that several proteins identified as associated with the TORC1 are important for nitrogen starvation-dependent diploid pseudohyphal growth. In contrast, invasive growth due to other nutritional stresses was generally not affected in mutant strains of these TORC1-associated proteins. Our studies suggest a role for TORC1 in yeast differentiation upon nitrogen starvation. Our studies also suggest the CEN.PK strain background of S. cerevisiae may be particularly useful for investigations of nitrogen starvation-induced diploid pseudohyphal growth.     

Induction of S. cerevisiae filamentous differentiation by slowed DNA synthesis involves Mec1, Rad53 and Swe1 checkpoint proteins

A key question in eukaryotic differentiation is whether there are common regulators or biochemical events that are required for diverse types of differentiation or whether there is a core mechanism for differentiation. The unicellular model organism Saccharomyces cerevisiae undergoes filamentous differentiation in response to environmental cues. Because conserved cell cycle regulators, the mitotic cyclin-dependent kinase Clb2/Cdc28, and its inhibitor Swe1 were found to be involved in both nitrogen starvation- and short chain alcohol-induced filamentous differentiation, they were identified as components of the core mechanism for filamentous differentiation. We report here that slowed DNA synthesis also induces yeast filamentous differentiation through conserved checkpoint proteins Mec1 and Rad53. Swe1 and Clb2 are also involved in this form of differentiation, and the core status of Swe1/Clb2/Cdc28 in the mechanism of filamentous differentiation has therefore been confirmed. Because the cAMP and filamentous growth mitogen-activated protein kinase pathways that mediate nitrogen starvation-induced filamentous differentiation are not required for slowed DNA synthesis-induced filamentous growth, they can therefore be excluded from the core mechanism. More significantly, slowed DNA synthesis also induces differentiation in mammalian cancer cells, and such stimulus conservation may indicate that the core mechanism for yeast filamentous differentiation is conserved in mammalian differentiation.     

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog.

Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein alpha subunit homologs in yeast, regulates pseudohyphal differentiation. Deltagpa2/Deltagpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2(Gly19Val) mutant or exogenous cAMP suppresses the Deltagpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels.     

The Morphogenesis Checkpoint in Saccharomyces cerevisiae: Cell Cycle Control of Swe1p Degradation by Hsl1p and Hsl7p

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.     

Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).