GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway.

The yeast RAS1 and RAS2 genes appear to be involved in control of cell growth in response to nutrients. Here we show that this growth control also involves a signal mediated by the heterotrimeric G protein alpha subunit homolog encoded by GPA2. A GPA2 null allele conferred a severe growth defect on cells containing a null allele of RAS2, although either mutation alone had little effect on growth rate. A constitutive allele of GPA2 could stimulate growth of a strain lacking both RAS genes. Constitutive GPA2 conferred heat shock sensitivity on both wild-type cells and cells lacking RAS function, but had no effect in a strain containing a null allele of SCH9, which encodes a kinase related to protein kinase A. The GPR1 gene was isolated and was found to encode a protein with the characteristics of a G protein-coupled receptor. Double Deltagpr1 Deltaras2 mutants displayed a severe growth defect that was suppressed by expression of the constitutive allele of GPA2, confirming that GPR1 acts upstream of GPA2. Gpr1p is expressed on the cell surface and requires sequences in the membrane-proximal region of its third cytoplasmic loop for function, as expected for a G protein-coupled receptor. GPR1 RNA was induced when cells were starved for nitrogen and amino acids. These results are consistent with a model in which the GPR1/GPA2 pathway activates the Sch9p kinase to generate a response that acts in parallel with that generated by the Ras/cAMP pathway, resulting in the integration of nutrient signals.     

Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism

Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.     

Saccharomyces Cerevisiae S288c Has a Mutation in Flo8, a Gene Required for Filamentous Growth

Diploid strains of baker's yeast Saccharomyces cerevisiae can grow in a cellular yeast form or in filaments called pseudohyphae. This dimorphic transition from yeast to pseudohyphae is induced by starvation for nitrogen. Not all laboratory strains are capable of this dimorphic switch; many grow only in the yeast form and fail to form pseudohyphae when starved for nitrogen. Analysis of the standard laboratory strain S288C shows that this defect in dimorphism results from a nonsense mutation in the FLO8 gene. This defect in FLO8 blocks pseudohyphal growth in diploids, haploid invasive growth, and flocculation. Since feral strains of S. cerevisiae are dimorphic and have a functional FLO8 gene, we suggest that the flo8 mutation was selected during laboratory cultivation.     

Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS.

Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.     

白念珠菌CaPPEl的克隆及其在酿酒酵母形态发生中的功能研究

白念珠菌的致病性与其形态转变相关,白念珠菌的形态转换受各种外界信号和细胞内信号转导途径的调控。转录因子Flo8在酿酒酵母形态发生中起重要作用,我们将白念珠菌基因组文库导入flo8缺失株中,筛选能够校正flo8缺失株侵入生长缺陷的基因,分离得到一个与酿酒酵母蛋白磷酸酯酶甲基酯酶PPEl同源的基因,命名为CaPPEl。CaPPEl的基因编码区全长1083bp,推测编码一个361氨基酸的蛋白。在单倍体酿酒酵母中,CaPPEl基因的表达可以部分回复flo8缺失株的侵入生长缺陷,但是在MAPK途径缺失株中不能进行侵入生长。在双倍体酿酒酵母中,CaPPEl基因的表达可以部分激活MAPK途径成员缺失株的菌丝生长缺陷,但却只能在flo8缺失株中产生微弱的激活作用。结果表明CaPpel在酿酒酵母的假菌丝生长和侵入生长中参与的信号转导途径不同。     

白念珠菌CaPPE1的克隆及其在酿酒酵母形态发生中的功能研究

白念珠茵的致病性与其形态转变相关,白念珠茵的形态转换受各种外界信号和细胞内信号转导途径的调控。转录因子Flo8在酿酒酵母形态发生中起重要作用,我们将白念珠茵基因组文库导入flo8缺失株中,筛选能够校正flo8缺失株侵入生长缺陷的基因,分离得到一个与酿酒酵母蛋白磷酸酯酶甲基酯酶PPEI同源的基因,命名为CaPPEl。CaPPEl的基因编码区全长1083bp,推测编码一个361氨基酸的蛋白。在单倍体酿酒酵母中,CaPPE1基因的表达可以部分回复flo8缺失株的侵入生长缺陷,但是在MAPK途径缺失株中不能进行侵入生长。在双倍体酿酒酵母中,CaPPEl基因的表达可以部分激活MAPK途径成员缺失株的茵丝生长缺陷,但却只能在flo8缺失株中产生微弱的激活作用。结果表明CaPpel在酿酒酵母的假茵丝生长和侵入生长中参与的信号转导途径不同。     

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.     

The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis

Saccharomyces cerevisiaepresents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions. To identify genes involved in cell wall morphogenesis, a haploid S. cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized. S. cerevisiaeW27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains. The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain. This produced an increase in the amount of proteins linked covalently to chitin. The same protein species, however, were released from the cell walls of the mutant and the parental strain. The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF. Transformation of S. cerevisiaeW27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype. These results suggest that the SRD2gene acts as a negative regulator of pseudohyphal growth.     

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.     

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.