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Vol. 18, Issue 9, 3237-3249, September 2007
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subunits in Cryptococcus neoformans
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710
Submitted February 16, 2007;
Revised June 5, 2007;
Accepted June 12, 2007
Monitoring Editor: Charles Boone
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits, most filamentous ascomycetes and basidiomycetes have three G subunits. Previous studies have defined the G subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which G subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two G subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional G into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 
, and 
subunits. GPCRs comprise the largest superfamily of cell surface receptors, responding to a panoply of extracellular stimuli as diverse as hormones, neurochemicals, odorants, nutrients, and light (Bockaert and Pin, 1999
). G proteins can bind to and hydrolyze GTP and alternate between the GTP-bound "on" and the GDP-bound "off" states. They function as molecular switches in signal transduction pathways to elicit diverse physiological processes. Ligand activation of GPCRs triggers the coupled G subunit to release GDP and bind to GTP, transforming them into the G-GTP active state. This results in a conformational change of the G subunit, which promotes dissociation from the G complex. The liberated G and G subunits may then interact with downstream effectors to switch on or off signaling cascades.
Two major signal transduction pathways, one responding to pheromone and the other to nutrients, are both governed by G proteins in Saccharomyces cerevisiae (Lengeler et al., 2000; Harashima and Heitman, 2004). These two pathways, the pheromone response pathway and the cAMP pathway, are among the most well understood signaling pathways in eukaryotes. Studies of this model have enormously contributed to the understanding of the mechanisms of G protein signaling and regulation. The G protein subunit Gpa1 is activated when pheromone binds to the pheromone receptors Ste2 or Ste3; the GTP-bound Gpa1 releases the G complex (Ste4/Ste18) to allow activation of their downstream effectors, including Ste20, Far1/Cdc24, and Ste5 to initiate mating responses (Dohlman and Thorner, 2001). In the cAMP-protein kinase A (PKA) pathway, the sugar receptor Gpr1 senses glucose and activates Gpa2 to stimulate adenylyl cyclase and increase intracellular cAMP levels, leading to PKA activation (Kubler et al., 1997; Lorenz and Heitman, 1997; Kraakman et al., 1999; Lorenz et al., 2000).
The GPCR and G protein (GPCR-G) repertoire in S. cerevisiae is extremely simple; only three GPCRs (Ste2/3 and Gpr1) and two G protein subunits (Gpa1 and Gpa2) are expressed. Many higher eukaryotes have an enormous complexity of GPCRs signaling. For example, 57 GPCRs and 14 G subunits are encoded in the slime mold Dictyostelium discoideum genome, and these numbers are further expanded to hundreds and even thousands for GPCRs and more than 20 G subunits in the nematode and human, respectively (Simon et al., 1991; Jansen et al., 1999; McCudden et al., 2005). As a consequence, it is a considerable challenge to study GPCR-G protein coupling specificity in these higher organisms, even though the number of expressed GPCRs and G proteins in each cell type may be more limited.
Several fungal genomes that have been annotated thus far reveal that the GPCR-G protein signaling networks in the filamentous ascomycetes and basidiomycetes are more complex than the S. cerevisiae paradigm, but not yet as extreme as that found in the multicellular eukaryotes. For example, in Neurospora crassa 10 potential GPCRs and 3 G subunits are present in the genome, and in A. nidulans there are 16 GPCRs and 3 G subunits (Borkovich et al., 2004; Yu, 2006). In the basidiomycete human pathogen Cryptococcus neoformans, 
31 genes are predicted to be potential GPCRs and 3 G subunits were identified (Loftus et al., 2005; Xue et al., 2006). Furthermore, the fungal G proteins do not fall into discrete G protein categories; they all are most closely related to Gi mammalian proteins, based on sequence homology. Thus, these fungal species, with more complicated G protein signaling circuits than S. cerevisiae, yet still genetically tractable, can serve as models with which to dissect GPCR- and G protein–coupling specificity.
Among the three G protein subunits expressed in C. neoformans (Gpa1, Gpa2, and Gpa3), only Gpa1 has a defined function. Genetic and biochemical analyses established that Gpa1 functions upstream of adenylyl cyclase and PKA and is required for pathogenesis and development of C. neoformans (Alspaugh et al., 1997, 2002; D'Souza et al., 2001). In a more recent study, the GPCR Gpr4 was shown to interact with Gpa1 and in response to methionine binding, Gpa1 initiates cAMP-PKA signaling (Xue et al., 2006). Conversely, it has been uncertain which G protein is coupled to the pheromone receptor, because neither of the other two G protein subunit null mutants (gpa2 or gpa3) exhibits a sterile phenotype. In fact, except in the budding and fission yeast, which only have two G subunits, no studies have conclusively shown the coupling/interactions between the pheromone receptors and the G subunits in any other fungus. In contrast, several G subunits have been identified that signal through the cAMP-PKA pathway in both ascomycetes and basidiomycetes (Regenfelder et al., 1997; Kruger et al., 1998; Kays et al., 2000; Shimizu and Keller, 2001; Han et al., 2004; Maidan et al., 2005).
Basic principles of G protein signaling and regulation in S. cerevisiae were elucidated over a decade ago, yet recently several unexpected facets were revealed. First, Gpa1 was for a long time considered to solely negatively regulate pheromone signaling by sequestering the G complex, but Slessareva et al. (2006) demonstrated that GTP-bound Gpa1 transduces a positive signal leading to mating events via the phosphatidylinositol 3-kinase Vps34 located on endosomal membranes. Second, a recent study showed that the RGS (regulator of G protein signaling) protein Sst2, which functions as a GAP for the G subunit Gpa1 to desensitize cells from pheromone signaling, binds to the activated pheromone receptor via its DEP domain (Ballon et al., 2006). This interaction localizes Sst2 in close proximity to its target Gpa1, presumably enhancing target specificity (Ballon et al., 2006). Similar to the expansion of G proteins found in the filamentous fungi, the RGS protein family has also expanded. For example, five distinct RGS proteins were identified in the A. nidulans genome (Yu, 2006). In C. neoformans, Crg1 was identified as an Sst2 homolog that functions to desensitize pheromone responses, but its target substrates are not yet well understood (Wang et al., 2004).
In this study, we dissected G protein signaling regulation of the pheromone response pathway in C. neoformans. Two G protein subunits, Gpa2 and Gpa3, were found to be critical to enable pheromone sensing and mating in haploid yeast cells. gpa2 gpa3 double mutants exhibit a cell–cell fusion defect and a bilateral sterile phenotype, indicating that both G protein subunits share an overlapping role in mating. However, Gpa2 promoted filamentous growth in its activated form, whereas dominant active Gpa3 inhibited mating. In addition to the G proteins, a G subunit Gpg2 was identified to be essential for mating. Furthermore, the novel RGS protein Crg2 was found to negatively regulate pheromone response during the early stages of mating and is required to complete sexual development in the terminal stage of mating. The pheromone receptor Ste3 interacts with both G subunits and RGS proteins in vivo.
Our study demonstrates that C. neoformans, and likely also in other fungal species that express more than two G subunits, additional G proteins are involved in the mating pathway regulatory circuits. We propose that this may be a consequence of the requirement for cells to choose their fate between budding yeast growth and asexual or sexual filamentous growth. Our studies also have implications for an understanding of the redundancy and specialized signaling roles of G protein–mediated GPCR signaling networks in multicellular eukaryotes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Fraser et al., 2003). Briefly, the 5' and 3' flanking regions of the target gene were amplified with gene-specific primers and the dominant NAT or NEO selectable markers were amplified with the universal M13F and M13R primers. These amplified 5' and 3' flanking regions, together with one of the dominant markers, were used as the DNA template for the second overlap PCR. The products of overlap PCR were purified and directly transformed into the cells by biolistic transformation (Davidson et al., 2000). Primers used for amplification of the 5' and 3' flanking regions of each gene disruption cassette are described in Supplementary Table 1.
Complementation of gpb1 and gpg2 Mutations
The wild-type GPB1 and GPG2 genes, including 1-kb upstream promoter and 0.5-kb downstream terminator regions were amplified with primer pairs JOHE17978/JOHE17979 and JOHE17947/JOHE17948 using wild-type genomic DNA as template. A second overlap PCR was conducted using the amplified wild-type GPB1 and GPG2 DNA fragment and the M13F/M13R amplified NEO marker as template with primers JOHE17978/M13R and JOHE17947/M13R. The overlap PCR products were purified and transformed into strains YPH312 and YPH272 to complement the gpb1 and gpg2 mutations. Southern analysis was performed to determine the copy number of the reintroduced wild-type alleles. Transformants with one copy of the wild-type GPB1 or GPG2 gene were analyzed phenotypically.
Double or Triple Deletion Mutant Generation
The serotype D MATa and MAT gpa2 gpa3 double mutants (YPH88 and YPH97) were isolated from the progeny of the cross MAT gpa2x MATa gpa3 (WSC26 x YPH86) and the serotype A MATa gpa2 gpa3 double mutant (YPH106) was also a progeny derived from the cross MAT gpa2x MATa gpa3 (YSB25 x YSB136). Basidiospores were micromanipulated according to methods described previously (Hsueh et al., 2006), and the genotype of each progeny was determined by PCR analyses. Serotype A MAT gpa2 gpa3 double mutant (YPH308) was created by deleting the GPA2 gene in the gpa3 mutant (YPH105) background using an overlap PCR and biolistic transformation approach. The crg1 gpa2 double mutants (YPH113 and YPH114) were progeny derived from the cross PPW196 (MATa crg1) x YSB25 (MAT gpa2) and crg1 gpa3 double mutants (YPH116 and YPH117) were progeny from the cross PPW111 (MAT crg1) x YSB136 (MATa gpa3). The crg1 gpa2 gpa3 triple mutants (YPH118 and YPH380) were progeny derived from the cross YPH114 (MAT crg1 gpa2) x YSB136 (MATa gpa3). The crg2 gpa2, crg2 gpa3, and crg2 gpa2 gpa3 mutants were isolated from the cross CDX50 (MAT crg2) x YPH106 (MATa gpa2 gpa3). Primer pairs JOHE6013/JOHE6014, JOHE8994/JOHE14774, JOHE8994/JOHE10923, and JOHE8994/JOHE12228 were used to identify the crg1, crg2, gpa2, and gpa3 mutant alleles, respectively. Sequences of these primers are listed in Supplementary Table 1.
Cell Fusion and Confrontation Assays
Cell fusion assays for gpa2, gpa3, and gpa2 gpa3 mutants were conducted with serotype D auxotrophic strains. To introduce a ura5 allele into the MAT gpa2, MAT gpa3, and MAT gpa2 gpa3 mutants, cells were grown on 5-FOA medium for 3–5 d, and spontaneous resistant colonies were selected and tested for their ability to grow on the SD-uracil medium. On the other hand, the MATa gpa2 lys1, MATa gpa3 lys1, and MATa gpa2 gpa3 lys1 strains were selected progeny from crosses YPH94 (MATa gpa2) x JEC31 (MAT lys1), YPH86 (MATa gpa3) x JEC31 (MAT lys1), and YPH88 (MATa gpa2 gpa3) x JEC31 (MAT lys1). Spores were isolated by micromanipulation, germinated, and analyzed by PCR and growth assays on SD-lysine medium.
To perform the cell fusion assays, MAT ura5 wild-type or mutant strains (JEC43, YPH130, WSC75, and YPH132) and MATa lys1 wild-type or mutant strains (JEC30, YPH95, YPH96, and YPH100) were grown in YPD liquid medium overnight. The next day, cells were collected, washed, and adjusted to 1 x 107 cells/ml. Equal numbers of cells of opposite mating type were mixed on V8 mating medium for 24 h in the dark. Cells were removed from the surface of medium and spread onto SD-uracil-lysine medium to select dikaryotic fusion products. The number of colonies from each cross was counted, and fusion efficiency calculated in proportion to wild type. To measure the fusion ability of the gpg2 mutant, equal numbers of strains YPH272 (MAT gpg2::NAT) and YSB121 (MATa NEO) were mixed and incubated on V8 mating medium for 24 h, and the mating colonies were harvested and spread on YPD medium containing nourseothricin and G418. The number of the colonies was determined and compared with that from the fusion between wild-type strains YSB119 (MATa NAT) and YSB121 (MATa NEO).
For confrontation assays, cells of opposite mating type were streaked in parallel, 3–4 mm apart, on V8 medium (pH 5) and incubated at 25°C in the dark for 3–4 d. Images were captured with a Nikon Eclipse E400 microscope equipped with a Nikon DXM1200F digital camera.
RNA Extraction and Northern Blot Analysis
Cells for RNA extraction were grown in YPD liquid culture overnight and the next day were harvested, washed, and adjusted to a density of 1 x 107 cells/ml. Drops of 5 µl of cells were spotted on V8 mating medium alone or with a mating partner for the nonmating and mating conditions, respectively. For both conditions, cells were incubated at 25°C for 24 h and harvested. RNA was extracted with TRIZOL Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Total RNA, 10 µg, was separated by denaturing agarose gel electrophoresis and blotted to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) and probed with [32P]dCTP-radiolabeled DNA fragments. Primers used for probe amplification are listed in Supplementary Table 2. Quantification of the expression signals was conducted with a Typhoon 9200 imager and Image Quantifier 5.2 software (Molecular Dynamics, Sunnyvale, CA).
Overexpression of Wild-Type and Dominant Active Gpa2Q203L and Gpa3Q206L Alleles
The dominant active GPA2Q203L and GPA3Q206L alleles were obtained by overlap PCR. Primers JOHE17654/JOHE17655 (for GPA2) and JOHE17656/JOHE17657 (for GPA3) were designed to introduce the Q-to-L substitution at the conserved position. The N-terminal half of the GPA2Q203L allele was amplified with primer pair JOHE17646 and JOHE17655, using H99 genomic DNA as template. The C-terminal half was amplified with primer pair JOHE17647 and JOHE17654. Primers JOHE17646 and JOHE17647 were then used for the second overlap PCR. By the same method, GPA3Q206L allele was amplified with primer pairs JOHE17648/JOHE17657 and JOHE17649/JOHE17656 (Supplementary Table 1). These amplified GPA2Q203L and GPA3Q206L alleles were digested with the restriction enzymes FseI and PacI and cloned into pXL1 driven by the constitutively expressed GPD1 promoter to generate the GPA2Q203L and GPA3Q206L overexpression plasmid YPP34 and YPP35. These plasmids were sequenced to confirm that the Q-to-L mutation (and no extraneous mutations) was present and then transformed into different backgrounds including wild-type, gpb1, and ste7 mutants. The transformants were analyzed with Northern analysis to determine the abundance of the GPA2Q203L and GPA3Q206L transcripts. Strains that expressed similar levels of GPA2Q203L and GPA3Q206L message were subjected to further -galactosidase activity assays and morphological analyses. Wild-type GPA2 and GPA3 alleles were amplified with primers JOHE17646/JOHE17647 and JOHE17648/JOHE17649, respectively. The DNA was digested with FseI and PacI and cloned into plasmid pXL1 driven by the constitutively expressed GPD1 promoter, resulting in the GPA2 and GPA3 overexpression constructs YPP44 and YPP47. These two constructs were transformed into strain JEC21 and the transformants that expressed a high abundance of GPA2 or GPA3 transcripts as detected by Northern analysis were further analyzed for their mating ability.
Split-Ubiquitin Protein–Protein Interaction Assays
The split-ubiquitin system was utilized to investigate the potential interactions between pheromone receptor Ste3 and G protein subunits Gpa1, Gpa2, and Gpa3, as well as interactions between Ste3 and RGS proteins Crg1 and Crg2. Vectors and yeast strains were included in the DUALmembrane Kit 2 (Dualsystem Biotech, Zurich, Switzerland). STE3 full-length cDNA was cloned into pCCW (the C-terminal half of the ubiquitin Cub protein was fused to the C-terminus of STE3). CRG1, GPA1, GPA2, and GPA3 full-length cDNAs were cloned into the pDL2XN vector (the mutated N-terminal half of ubiquitin NubG protein was fused to the test proteins' C-termini). CRG2 full-length cDNA was cloned into the pDSL-NX vector (the NubG protein was fused to the N-terminus of CRG2). All cDNA sequences were confirmed by DNA sequencing. Cub and NubG fusion constructs were cotransformed into host yeast strain NMY32. Interaction was determined by the growth of yeast transformants on medium lacking histidine and also by measuring -galactosidase activity. Primers used for the generation of the fusion alleles are listed in Supplementary Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits are expressed in C. neoformans. Gpa2 and Gpa3 were identified in the genome sequence and share homology with S. cerevisiae Gpa1 (GenBank Accession numbers AAQ62550 and AAQ74379). Gpa2 and Gpa3 are 45% identical to each other, and both have the conserved GTPase motif and N-terminal Met-Gly sequence and an adjacent Cys residue that are required for myristoylation and palmitoylation. To identify which G protein subunit governs the pheromone response pathway, gpa2 and gpa3 null mutants were generated in both a and cells in the C. neoformans var. grubii (serotype A) and var. neoformans (serotype D) backgrounds. Surprisingly, neither the gpa2 or gpa3 single mutants exhibited mating defects, even in bilateral crosses, suggesting that cells can sense pheromone without either G subunit (Figure 1, A and B). We also considered and excluded the hypothesis that Gpa2 and Gpa3 might play cell type–specific roles in mating (i.e., gpa2x a gpa3 and gpa3x a gpa2 crosses were fertile). These results led us to hypothesize that the two G subunit might be functionally redundant in pheromone sensing or that no G subunits regulate mating.
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subunits were isolated and examined for their mating properties. In unilateral crosses (mutant x wild-type), the gpa2 gpa3 mutants were still fertile in both mating types and serotypes. Sexual structures, including dikaryotic filaments, fused clamp cells, basidia, and basidiospores were all observed, but were less abundant when compared with a wild-type cross (Figure 1, A and B). Similarly, gpa2 gpa3 double mutants are still fertile when crossed to gpa2 or gpa3 single mutants (Supplementary Figure 1). Nonetheless, in the bilateral cross (mutant x mutant), gpa2 gpa3 double mutants exhibited severe mating defects. No mating structures were observed in serotype A mutants, whereas in serotype D mutants, because of their self-filamentous phenotype (Figure 2A), filaments and basidia were still observed but no basidiospores (meiotic progeny) were produced (Figure 1B). DAPI staining showed that filaments produced in the serotype D gpa2 gpa3 double mutant bilateral cross were monokaryotic instead of dikaryotic, demonstrating that these hyphae are not mating structures (Figure 1B).
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gpa2
::NAT gpa3::NEO double mutant was crossed to the wild-type MATa strain KN99a, and 42 basidiospores were isolated. Among these progeny, 12 wild-type, 10 gpa2::NAT single mutant, 16 gpa3::NEO single mutant, and 4 gpa2::NAT gpa3::NEO double mutants were found. The wild-type progeny of different mating types were crossed to each other, and all crosses were fertile. Thus, the sterile phenotype displayed by gpa2 gpa3 double mutants is not attributable to a background mutation. On the other hand, when the four gpa2::NAT gpa3::NEO double mutants of opposite mating types were crossed to each other or to the parental gpa2 gpa3 mutants, all exhibited a bilateral sterile phenotype. Thus, the bilateral sterile phenotype is linked to the gpa2 and gpa3 mutations.
The bilateral sterile phenotype of the gpa2 gpa3 mutant strongly suggests that both G subunits play a role in the mating pathway, and the double mutants are likely to be insensitive to the pheromone secreted by the mating partner. One of the first steps during sexual development in Cryptococcus involves the fusion of a and cells to form a heterokaryon, and defects in pheromone sensing compromise this process. To determine whether the sterile phenotype of a gpa2 gpa3 mutant is caused by an inability to undergo cell–cell fusion, quantitative cell–cell fusion assays were performed using different auxotrophic strains (see Materials and Methods). In a gpa2 gpa3 double mutant bilateral cross, the fusion efficiency is dramatically reduced by at least 105 fold (0.0008% compared with wild-type; 100%). In unilateral mutant crosses, the MATa gpa2 gpa3 mutant fused to wild-type cells at a level of 90% efficiency and the MAT gpa2 gpa3 double mutant fused 51% as efficiently as wild type (Figure 1 B, bottom panel). This analysis demonstrates that if both a and cells are insensitive to pheromone, they are unable to fuse with each other; however, if pheromone sensing is only perturbed in one of the two mating partners, a substantial proportion of the cells are still capable of completing the sexual cycle. This genetic evidence supports the hypothesis that both Gpa2 and Gpa3 are involved in pheromone sensing and are at least in part functionally redundant.
gpa3 Deletion Leads to a Constitutively Active Pheromone Response
In S. cerevisiae, one major function of the G subunit Gpa1 is to sequester the G complex to prevent pheromone signaling under nonmating conditions (Nomoto et al., 1990). As a consequence, the pheromone response pathway is constitutively activated in gpa1 mutant cells in which the G complex is liberated. In C. neoformans, an analogous positively acting G subunit, Gpb1, has been characterized previously (Wang et al., 2000). Thus, we hypothesized that a constitutively active pheromone response would be observed when the Gpb1-interacting G subunit was removed.
One prominent phenotype of the gpa3 mutant in the serotype D background (but not the serotype A background) was dramatically enhanced self-filamentation. After 48-h incubation on V8 medium, prolific filamentation was observed along the peripheries of gpa3 mutant colonies, whereas in the wild-type strain, filamentation was only observed after 2 wk. This hyperfilamentation phenotype is dependent on the G subunit Gpb1 because gpa3 gpb1 double mutants were nonfilamentous and reintroduction of the GPB1 gene (gpa3 gpb1 GPB1, Figure 2A, right panel) complemented this defect and restored filamentous growth. The hyperfilamentous phenotype of gpa3 cells does not require Gpa2 as the gpa2 gpa3 double mutant showed a similar phenotype as the gpa3 single mutant (Figure 2A). DAPI staining showed that the hyphal cells produced by gpa3 single or gpa2 gpa3 double mutants contained a single nucleus, unfused clamp cells, and short basidiospore chains, all of which are hallmarks of monokaryotic fruiting (Figure 2B). Fruiting is a same-sex mating process that involves cells of only one mating type, and so far has only been routinely observed in serotype D strains (Lin et al., 2005). Several components that function in the pheromone signaling have been shown to contribute to this process (Shen et al., 2002; Davidson et al., 2003). Interestingly, the monokaryotic hyphae produced by MAT gpa3 and MAT gpa2 gpa3 double mutants were more prolific than MATa gpa3 or gpa2 gpa3 strains, indicating that the MAT locus may play a role in filamentous growth (Figure 2C). Indeed, it was recently shown that the MAT allele serves as one of several quantitative trait loci that promote filamentation (Lin et al., 2006).
We suspected that the hyperfilamentation phenotype of the gpa3 mutant was a result of a constitutively active pheromone response because it has been shown that overexpression of the pheromone genes enhances monokaryotic fruiting (Shen et al., 2002). Thus, the abundance of pheromone transcripts in the wild-type, gpa2, gpa3, or gpa2 gpa3 double mutant cultured alone or with a mating partner on V8 medium was determined. As shown in Figure 3, in wild-type cells, the pheromone genes were expressed at a basal level in monoculture and were induced during mating coculture conditions (Figure 3). In contrast, gpa3 or gpa2 gpa3 double mutant cells exhibited a constitutively active pheromone response; in both a and cell types, cultured alone, the expression of the pheromone genes was dramatically induced. Gpb1, the G protein subunit required for mitogen-activated protein kinase (MAPK) cascade activation of the pheromone response pathway, was also induced under mating conditions. Quantification using a phosphorimager documented that the GPB1 transcripts were 1.6-fold more abundant during mating, whereas the expression of GPB1 in the gpa3 or gpa2 gpa3 double mutant was 1.3–1.8-fold higher than in wild-type cells, even in the absence of a mating partner (Figure 3). The expression pattern of the MF1 and MFa1 pheromone genes and GPB1 demonstrates that in the gpa3 and gpa2 gpa3 double mutants, the pheromone signaling pathway is engaged without the prerequisite of pheromone activation. These results suggest that although Gpa2 and Gpa3 play redundant roles in pheromone sensing, Gpa3 has a prominent function in repressing pheromone signaling via sequestration of Gpb1 in nonmating cells.
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subunits play overlapping roles in pheromone sensing. However, deletion of GPA3 leads to a constitutively active pheromone response, whereas deletion of GPA2 does not, suggesting they serve different roles during C. neoformans sexual development. To further investigate how these two G protein subunits are regulated, the same RNA blot that was probed for pheromone gene transcripts was used to analyze GPA2 and GPA3 expression. In wild-type cells, growth alone on V8 mating medium induced GPA3 expression, and no further induction was apparent following exposure to an opposite mating type partner. In contrast, the GPA2 transcripts were undetectable in monoculture and were strongly induced by the opposite mating partner (Figure 3). The GPA2 expression pattern is quite similar to that of the MF/MFa pheromone genes. In addition, in the gpa3 mutants, in which the pheromone response pathway is constitutively activated, even without pheromone stimulation from opposite mating-type cells, GPA2 was also induced (Figure 3). This demonstrates that GPA2 expression is specifically triggered by pheromone signaling pathway activation. Therefore, the expression of GPA2 and GPA3 is temporally regulated during mating in response to different cues. Cells initially express GPA3 in response to signals present in the mating medium and GPA2 is subsequently induced when the mating partner is encountered.
Two RGS Proteins, Crg1 and Crg2, Negatively Regulate Pheromone Responses
The RGS protein Crg1 has been previously characterized as a negative regulator of the pheromone response pathway in C. neoformans; cells that lack Crg1 are hypersensitive to pheromone, resulting in the formation of conjugation tubes observed in a confrontation assay (Nielsen et al., 2003; Wang et al., 2004). To determine which G subunit Crg1 acts on, we generated crg1 gpa2, crg1 gpa3 double mutants and crg1 gpa2 gpa3 triple mutants in both mating type backgrounds to examine whether the crg1 phenotype is dependent on either or both G subunits. These strains were isolated in the serotype A background in order to circumvent the self-filamentous phenotype of the serotype D gpa3 or gpa2 gpa3 double mutants.
As shown in Figure 4A, in confrontation assays both crg1 gpa2 and crg1 gpa3 double mutants could still produce conjugation tubes in response to cells of the opposite mating-type, indicating that both are capable of generating and responding to pheromones. In confrontation assays the crg1 gpa2 gpa3 triple mutant still secreted pheromones (based on the formation of conjugation tubes by the crg1 partner), but they failed to sense pheromone and did not produce conjugation tubes (Figure 4A). These findings indicate that the pheromone hypersensitive phenotype of crg1 mutants requires Gpa2 or Gpa3 in cells of both mating types.
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subunit, CRG2 was deleted in the wild-type serotype A background, and the resulting crg2 mutants were phenotypically analyzed. The most prominent phenotype for a crg2 mutant was enhanced filamentation in mating that was readily observed macroscopically (Figure 4B). After coculturing wild-type a and cells on V8 mating medium, filaments at the edge of the colony were apparent after 5 d, whereas in a crg2 unilateral cross, filaments were much more prolific and could be observed as early as 48 h of coculture. This phenotype argues that Crg2, like Crg1, negatively regulates the pheromone response pathway by desensitizing the G subunits. Although filamentation was accelerated and enhanced in the crg2 mutant, sporulation was impaired and most basidia bore only a few spores, in contrast to the four long spore chains observed in a wild-type cross. This result suggests that Crg2 may play additional positive roles at the later stages of mating. To dissect the genetic relationship between Crg2 and Gpa2 and Gpa3, double and triple mutants were generated and analyzed for mating. As shown in Figure 4B, the hyperfilamentation phenotype of crg2 is dependent on the presence of either one of the two G subunits and a crg2 gpa2 gpa3 triple mutant exhibiting mating defects, resembling the phenotype of a gpa2 gpa3 double mutant.
The expression of both CRG1 and CRG2 was examined in wild-type, gpa2, gpa3, and gpa2 gpa3 double mutant cells in the presence or absence of a mating partner. CRG1 transcripts were detected in the gpa3 and gpa2 gpa3 double mutant monocultures and also in wild-type cells during mating (Figure 3). This pattern indicates that the expression of CRG1 is pheromone responsive, similar to the regulation of the pheromone genes and GPA2. CRG2 transcripts, on the other hand, were present in cells before mating, and their abundance did not increase when cells were exposed to a mating partner (Figure 3). Therefore, like GPA3, CRG2 was expressed at a constant basal level irrespective of pheromone signaling. These analyses revealed that the expression of the G subunits and RGS proteins are regulated coordinately (CRG2 like GPA3, and CRG1 like GPA2). Interestingly, genetic analysis revealed that CRG1 is linked to GPA2. In an gpa2x a crg1 cross, only 2 of 84 progeny analyzed were wild-type or gpa2 crg1 mutant. This suggested that the two genes are 2.4 cm apart, and analysis of the completed serotype A and D genomes revealed that they are separated by a 40-kb interval.
To provide further evidence to support the idea that both Crg1 and Crg2 are involved in the pheromone response pathway, we analyzed pheromone gene expression in the wild-type and mutant strains. Similar to findings shown in Figure 3, the expression of the pheromone gene was elevated under mating conditions and in the gpa3 mutant cultured alone (Figure 4C). The level of induction was lower in the serotype A strains (Figure 4C) than that observed in serotype D strains (Figure 3), in accord with the more proficient mating of serotype D strains compared with serotype A strains in the laboratory. On the other hand, the pheromone transcripts were 4.6- and 7.3-fold, respectively, more abundant than wild-type in the crg1 and crg2 mutant crosses (Figure 4C). This indicates that cells lacking either of the RGS proteins fail to desensitize the pheromone response. However, it remains to be determined if the elevated pheromone expression in crg2 mutants is an indirect consequence of Gpa1 activation.
Dominant Active GPA2Q203L and GPA3Q206L Alleles Display Opposing Signaling Capacities
Studies in S. cerevisiae showed that overexpression of a dominant active (GTPase-deficient) Gpa1 mutant led to a subset of the mating responses induced by Ste4-Ste18, suggesting that in addition to sequestering the G complex, Gpa1 positively signals to the pheromone response pathway (Metodiev et al., 2002; Guo et al., 2003). Recent studies further demonstrated that the GTP-bound Gpa1 translocates onto endosomes, activates phosphatidylinositol 3-kinase, and enhances pheromone responsiveness of the cell (Slessareva et al., 2006).
To investigate whether C. neoformans Gpa2 and Gpa3 have any intrinsic signaling roles, dominant active GPA2Q203L and GPA3Q206L alleles were overexpressed under the control of a constitutive GPD1 promoter in a strain that also bears an MF1 pheromone promoter-driven -galactosidase reporter gene. Cells that overexpressed GPA2Q203L were self-filamentous on V8 mating medium and had 4.2-fold higher -galactosidase activity compared with wild-type cells (Figure 5A). The -galactosidase activity in the GPA2Q203L strain in the absence of a mating partner was similar to the level of wild-type cells under mating conditions, indicating that a mating response was induced in GPA2Q203L cells alone. In the presence of a mating partner, overexpression of GPA2Q203L elicited a higher level of -galactosidase activity than wild type, and these cells mated more robustly and prolific filamentation was observed (Figure 5A). In contrast, the GPA3Q206L allele exhibited an opposing signaling activity. In the absence of a mating partner on V8 medium, overexpression of GPA3Q206L conferred no difference in -galactosidase activity compared with wild-type cells. However, during mating, when pheromone expression was highly elevated in wild-type cells, it was inhibited in cells expressing the GPA3Q206L allele; as a consequence, mating ability was diminished and less filamentation was observed (Figure 5A).
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complex, fine tuning can be achieved via the G subunits. Gpa2Q203L positively regulates mating responses, whereas Gpa3Q206L inhibits mating. This result prompted us to examine in more detail the pheromone expression pattern in the gpa2 mutant, in which the negative signaling role of Gpa3 would be unopposed. As seen in Figure 4C, pheromone expression was moderately decreased in the gpa2x gpa2 bilateral mutant cross when compared with wild-type in the serotype A strains. The Northern blot analysis was repeated to quantify the pheromone and actin signals. The result revealed that pheromone gene expression was 2.34-fold lower in the gpa2x gpa2 cross, indicating that pheromone induction during the early stages of mating was modestly diminished, although this difference was not observed in the serotype D strains in which mating is more robust (Figure 3). In accord with the decreased level of pheromone expression, serotype A gpa2x gpa2 crosses exhibited less robust filamentation when compared with a wild-type cross, which was in contrast to the enhanced mating observed in the gpa3x gpa3 cross (Figure 1 A, first panel).
To further determine whether the self-filamentous phenotype caused by GPA2Q203L overexpression required the G subunit or MAPK module, we overexpressed the GPA2Q203L allele in the gpb1 and ste7 mutant strains and monitored colony morphology on V8 mating medium. As shown in Figure 5B, in contrast to wild-type cells, gpb1 and ste7 mutant overexpressing GPA2Q203L were not self-filamentous, indicating that Gpb1 and Ste7 are needed to sustain GPA2Q203L signaling or that Gpa2-GTP signaling must occur in parallel with the MAPK cascade. Furthermore, it shows that both Gpa2-GTP and Gpb1 function coordinately to active MAPK signaling. This requirement was also seen for Gpa1 signaling in S. cerevisiae (Guo et al., 2003) and suggests that the regulation of a positive signal evoked by the dominant active G mutant is evolutionarily conserved.
Pheromone Receptor Ste3 Interacts with Both Gpa2 and Gpa3
Genetic analysis demonstrates that both Gpa2 and Gpa3 participate in the Cryptococcus mating pathway and a double mutant loses the ability to sense pheromone from the mating partner. We therefore hypothesized that the pheromone receptor Ste3/a is likely coupled to both G protein subunits, which then transmit signals to the downstream MAPK cascade to control genes involved in mating.
To corroborate this hypothesis, we used the split-ubiquitin two-hybrid system that was developed to assess physical interactions for membrane-associated proteins in a physiological setting (Stagljar et al., 1998). The C-terminal fragment of ubiquitin carrying the artificial transcription factor LexA-VP16 (Cub) was fused to a bait protein and the mutated N-terminal fragment (NubG) was fused to a prey. Protein–protein interactions enable reassociation between the two halves of split ubiquitin, resulting in cleavage and release of LexA-VP16, which then activates the HIS3, ADE2, and lacZ nuclear reporter genes. In our assays, the Cub domain was fused to the C-terminus of the pheromone receptor Ste3, and the NubG domain was fused to Gpa1, Gpa2, or Gpa3. The Ste3::Cub plasmid was cotransformed with one of the G-NubG plasmids, and as shown in Figure 6, cells that coexpressed Ste3::Cub and Gpa2::NubG, or Ste3::Cub and Gpa3::NubG, grew on medium lacking histidine, whereas cells coexpressing Ste3::Cub and Gpa1::NubG failed to grow. This finding indicates that the pheromone receptor Ste3 interacts with both Gpa2 and Gpa3, but not Gpa1 in vivo. The magnitude of the interactions was further quantified by -galactosidase activity assays (Figure 6). Compared with the positive control, the interactions between the pheromone receptor and both G subunits were more moderate. In accord with this result, cells coexpressing Ste3 and Gpa2 or Gpa3 could not grow on medium lacking adenine, consistent with a less stable or transient interaction.
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). Crg1, like Sst2, also possesses a DEP domain; Crg2, although it does not carry a DEP domain, has three predicted C-terminal transmembrane helices. Therefore, we hypothesized that both RGS proteins have the potential to bind to pheromone receptors. To examine this hypothesis, the split-ubiquitin assay was used to determine if Ste3 and Crg1/Crg2 interact. As shown in Figure 6, Crg1 displayed a stronger interaction in the split ubiquitin assay with the pheromone receptor Ste3 compared with Crg2. Cells that coexpressed Ste3::Cub and Crg1::NubG or Crg2::NubG both grew on medium lacking histidine. -galactosidase activity assays, however, revealed that Crg1 yielded a stronger signal with the pheromone receptor Ste3. We therefore conclude that similar to what was seen in S. cerevisiae, Crg1 is in a complex that also contains Ste3, likely through the DEP domain and Crg2, which is membrane-bound, also interacts with the pheromone receptor, possibly with a lower affinity.
G Subunit Gpg2 Is Essential for Sexual Development
The G protein subunit Gpb1 activates the MAPK cascade to trigger mating responses in C. neoformans similar to the signaling cascade in S. cerevisiae, but the Gpb1-interacting G subunit has been elusive (Wang et al., 2000). A putative open reading frame (ORF) annotated in the genome of the sequenced serotype D strain B3501 (CNBL0880) was found to share similarities with known fungal G subunits such as Ste18 in S. cerevisiae, including the hallmark C-terminal CAAX motif (Supplementary Figure 2). This gene, GPG2, was also independently identified by Palmer et al. (2006), and it was shown that Gpg2 physically interacts with Gpb1 in a yeast two-hybrid assay.
To investigate whether this gene indeed encodes a G subunit that activates mating responses via pheromone stimulation, the GPG2 gene was deleted in the wild-type serotype A MAT background, and the mutant was crossed to wild-type a cells. The gpg2 mutation completely abolished mating, even in a unilateral cross, a phenotype highly reminiscent of the gpb1 mutant (Figure 7). Based on a quantitative cell–cell fusion assay, it was determined that the gpg2 mutant was unable to fuse with wild-type a cells and hence was sterile (Figure 7). Complementation of the gpg2 mutation with the wild-type GPG2 allele restored the mating ability of the gpg2 mutant, demonstrating that this mating defect is attributable to the loss of GPG2 gene function (Figure 7). Thus, we conclude that Gpg2 is a subunit that is essential for pheromone signaling in C. neoformans.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit is coupled to the pheromone receptor and governs the pheromone signaling cascade in C. neoformans? Surprisingly, all lines of evidences indicate that both Gpa2 and Gpa3 are recruited into the signaling circuit, with both overlapping and specialized signaling activities.
These findings allow us to propose a model of the pheromone signaling network in C. neoformans (Figure 8). Compared with S. cerevisiae, in which only one G protein subunit (Gpa1) and an RGS protein (Sst2) are involved in pheromone signaling regulation, two G protein subunits Gpa2 and Gpa3 and two RGS proteins Crg1 and Crg2 were identified to regulate the mating pathway in C. neoformans. Under nutrient limiting conditions, cells in the serotype D lineage express Gpa3 to inhibit basal signaling and filamentous growth via sequestration of the G complex. Meanwhile, the RGS protein Crg2 functions to terminate signaling input from spontaneously activated Gpa3 and therefore, loss of either component leads to an activation of the pheromone response. When a mating partner is encountered, pheromone binding to the Ste3a/ receptor on the cell membrane triggers Gpa3 to dissociate from the Gpb1/Gpg2 complex, activating the MAPK signaling cascade and inducing the expression of pheromone-responsive genes, including GPA2, MF/a, and CRG1. After this first wave of pheromone induced-signaling, Gpa2, now in much higher abundance, may generate a second wave of signaling contributing to mating in its GTP-bound state. On the other hand, the RGS protein Crg1, a pheromone-inducible gene and a built-in feedback regulator, facilitates signal termination by shortening the lifetime of GTP-bound Gpa2 and desensitizes cells to pheromone secreted by the mating partner.
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; Davidson et al., 2003). One major factor that may contribute to this difference is the constitutive pheromone signaling in the gpa2 gpa3 mutants due to the release of G. In the ste11, ste7, and cpk1 mutants, pheromone signaling is blocked and cells exhibit a unilateral fusion defect; conversely, because the pheromone signaling is constitutively active in the gpa2 gpa3 mutants, they do not have the fusion defect in a unilateral cross.
The molecular mechanisms regarding how Gpa2 positively signals mating responses in C. neoformans, although unclear, are of considerable interest. Three models are proposed here. In the first model, Gpa2 binds to the Gpb1/Gpg2 complex and pheromone activation of Gpa2 triggers its dissociation from and both Gpa2-GTP and free activate downstream effectors to induce mating responses; this model is most analogous to pheromone signaling in S. cerevisiae. In accord with this hypothesis, we found that when GPA2 or GPA3 is overexpressed in the wild-type, mating is reduced (Supplementary Figure 3). This suggests that both G subunits may have the capacity to sequester the G complex. On the other hand, the higher expression levels of both of the pheromone genes and GPA2 in the gpa3 mutant background suggest alternative models. If Gpa2 indeed forms a complex with the Gpb1/Gpg2 complex, the highly expressed GPA2 is likely to restrain the availability of free in the gpa3 mutant and reduce activation of pheromone signaling. However, the pheromone genes are highly expressed in the gpa3 mutant. Thus, in the second and third model, we propose that Gpa2 could form a complex with other G like proteins or it could act as a solo G.
A central question is the identity of the downstream effectors of Gpa2. Studies in S. cerevisiae revealed that potential G effectors include the MAPK Fus3, an RNA-binding protein Scp160, and phosphatidylinositol 3-kinase Vps34 (Metodiev et al., 2002; Guo et al., 2003; Slessareva et al., 2006). These are candidate effectors of C. neoformans Gpa2 and, in addition, our results show that Gpa2 activity requires functional Gpb1 and Ste7. A recent report showed that a mammalian Go subunit can directly target PKA (Ghil et al., 2006). Thus, we do not exclude the possibility that Gpa2 may act via other signaling cascades, such as the cAMP-PKA pathway that also contributes to govern mating. Future investigations will be required to discover the downstream effectors of Gpa2.
Mating in C. neoformans is regulated by both pheromones and nutrients, and a principle role of Gpa3 is to inhibit filamentous growth in environments in which cells are prepared for mating but a mating partner is not present. Thus, Gpa3 is a negative regulator for the yeast to hyphal dimorphic transition. We propose that an analogy may exist with those human fungal pathogens that are dimorphic and able to switch between yeast and hyphal growth. Examples include Penicillium marneffei, Histoplasma capsulatum, and Coccidioides spp. One critical signal for this transition is temperature; cells grow as mycelia at room temperature and switch to yeast growth when temperature is elevated. Similarly, C. neoformans can only grow filamentously below 25°C. The gpa3 mutant is hyperfilamentous at permissive temperature (18–25°C), but it only grows as a yeast at 37°C. These dimorphic fungi all have three G protein subunits and are thought to have cryptic sexual cycles, and studies from P. marneffei show that one of the G subunits (GasA) functions as a negative regulator for asexual development (Borneman et al., 2001; Zuber et al., 2002). We hypothesize that in the dimorphic fungi, one of the G protein subunits may function analogously to Gpa3 in C. neoformans.
Functional parallels for the Gpa2/Gpa3 signaling paradigm may be found in other organisms. In chemotactic cells such as neutrophils and the slime mold D. discoideum, cell polarity and movement signaling is positively transmitted by G, and the G subunits may not be required (Wu et al., 1995; Neptune and Bourne, 1997; Neptune et al., 1999). However, more recent studies in both systems have suggested that G subunits are more than just passive components in the signaling networks (Brzostowski et al., 2002; Xu et al., 2003). In Dictyostelium, two G subunits (G2 and G9) were shown to couple to the cAMP receptor CAR1 and mediate cAMP chemotaxis. G2 is mainly responsible for the release of G, whereas G9 negatively regulates multiple downstream pathways activated by G (Brzostowski et al., 2004). Another analogy is evident in mammalian 2 adrenergic receptor (2-AR)-mediated signaling events. On agonist binding, 2-AR activates Gs to activate the cAMP-PKA pathway. Activated PKA then phosphorylates 2-AR and switches its coupling specificity to another class of G (Gi), which in turn activates MAPK via the released G (Daaka et al., 1997). In addition to 2-AR, PKA-mediated G protein-coupling switching is also observed with the prostacyclin receptor (Lawler et al., 2001). Through switches in G protein–coupling specificity, receptors can govern distinct signaling pathways and generate temporal cellular signaling waves.
Functional redundancy between G proteins has been previously described in some organisms. In the filamentous ascomycete N. crassa the G subunits Gna-1 and Gna-2 share overlapping functions in hyphal extension, growth under stress conditions, and female fertility (Baasiri et al., 1997; Kim and Borkovich, 2004). In C. elegans, two G subunits (GPA-16 and GOA-1) were shown to coregulate spindle position and orientation in embryos, and inactivation of both genes causes embryonic lethality (Gotta and Ahringer, 2001). Furthermore, like Gpa2 and Gpa3 in C. neoformans and in both N. crassa and the nematode, the above-mentioned G proteins share the highest level of similarity to each other and may have resulted from gene duplication events.
It is well understood that the budding yeast only requires one G (Gpa1) for mating; on the other hand, in C. neoformans, two G subunits with both overlapping and divergent functions are involved in sexual development. One of the subunits promotes mating, whereas the other inhibits filamentous growth, yet either one is sufficient to enable mating. Analyzing the number of G subunits present in most annotated fungal genomes suggests that one G subunit has been lost in the hemiascomycete lineage. This also suggests that the pattern of three G subunits is more ancient. We propose that the involvement of two G subunits in sexual development may be evolutionarily conserved among other fungi that express three different G proteins. The cell fate decision in S. cerevisiae is a simple binary choice; cells grow either as asexual budding yeasts, or they undergo mating when an appropriate partner is present. Conversely, C. neoformans and other dimorphic fungi have a ternary choice regarding their cell fate; they can grow as budding yeasts or switch to asexual or sexual filamentous development. The addition of an extra G subunit enables increased signaling complexity, which allows cells to make an appropriate ternary choice during the various stages of the life cycle. Finally, our study provides insights into GPCR and G protein–coupling specificity relevant to an understanding of similar signaling circuits operating in higher multicellular organisms, including slime molds, nematodes, flies, and even mammals.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Joseph Heitman (heitm001{at}duke.edu).
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alspaugh, J. A., Pukkila-Worley, R., Harashima, T., Cavallo, L. M., Funnell, D., Cox, G. M., Perfect, J. R., Kronstad, J. W., and Heitman, J. (2002). Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans. Eukaryot. Cell 1, 75–84.
Baasiri, R. A., Lu, X., Rowley, P. S., Turner, G. E., and Borkovich, K. A. (1997). Overlapping functions for two G protein alpha subunits in Neurospora crassa. Genetics 147, 137–145.[Abstract]
Ballon, D. R., Flanary, P. L., Gladue, D. P., Konopka, J. B., Dohlman, H. G., and Thorner, J. (2006). DEP-domain-mediated regulation of GPCR signaling responses. Cell 126, 1079–1093.[CrossRef][Medline]
Bockaert, J., and Pin, J. P. (1999). Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J 18, 1723–1729.[CrossRef][Medline]
Borkovich, K. A. et al. (2004). Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol. Mol. Biol. Rev 68, 1–108.
Borneman, A. R., Hynes, M. J., and Andrianopoulos, A. (2001). An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant. Genetics 157, 1003–1014.
Brzostowski, J. A., Johnson, C., and Kimmel, A. R. (2002). Galpha-mediated inhibition of developmental signal response. Curr. Biol 12, 1199–1208.[CrossRef][Medline]
Brzostowski, J. A., Parent, C. A., and Kimmel, A. R. (2004). A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement. Genes Dev 18, 805–815.
D'Souza, C. A., Alspaugh, J. A., Yue, C., Harashima, T., Cox, G. M., Perfect, J. R., and Heitman, J. (2001). Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol. Cell. Biol 21, 3179–3191.
Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997). Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature 390, 88–91.[CrossRef][Medline]
Davidson, R. C., Blankenship, J. R., Kraus, P. R., de Jesus Berrios, M., Hull, C. M., D'Souza, C., Wang, P., and Heitman, J. (2002). A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 2607–2615.
Davidson, R. C., Cruz, M. C., Sia, R. A., Allen, B., Alspaugh, J. A., and Heitman, J. (2000). Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genet Biol 29, 38–48.[CrossRef][Medline]
Davidson, R. C., Nichols, C. B., Cox, G. M., Perfect, J. R., and Heitman, J. (2003). A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans. Mol. Microbiol 49, 469–485.[CrossRef][Medline]
Dohlman, H. G., and Thorner, J. W. (2001). Regulation of G protein-initiated signal transduction in yeast: paradigms and principles. Annu. Rev. Biochem 70, 703–754.[CrossRef][Medline]
Fraser, J. A., Subaran, R. L., Nichols, C. B., and Heitman, J. (2003). Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot. Cell 2, 1036–1045.
Ghil, S., Choi, J. M., Kim, S. S., Lee, Y. D., Liao, Y., Birnbaumer, L., and Suh-Kim, H. (2006). Compartmentalization of protein kinase A signaling by the heterotrimeric G protein Go. Proc. Natl. Acad. Sci. USA 103, 19158–19163.
Gotta, M., and Ahringer, J. (2001). Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos. Nat. Cell Biol 3, 297–300.[CrossRef][Medline]
Guo, M., Aston, C., Burchett, S. A., Dyke, C., Fields, S., Rajarao, S. J., Uetz, P., Wang, Y., Young, K., and Dohlman, H. G. (2003). The yeast G protein alpha subunit Gpa1 transmits a signal through an RNA binding effector protein Scp160. Mol. Cell 12, 517–524.[CrossRef][Medline]
Han, K. H., Seo, J. A., and Yu, J. H. (2004). Regulators of G-protein signalling in Aspergillus nidulans: RgsA downregulates stress response and stimulates asexual sporulation through attenuation of GanB (Galpha) signalling. Mol. Microbiol 53, 529–540.[CrossRef][Medline]
Harashima, T., and Heitman, J. (2004). Nutrient control of dimorphic growth in Saccharomyces cerevisiae. In: Nutrient Induced Responses in Eukaryotic Cells, Vol. 7, ed. J. Winderickx and P.M. Taylor, Berlin, Germany: Springer-Verlag, 131–169.
Hsueh, Y. P., Idnurm, A., and Heitman, J. (2006). Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome. PLoS Genet 2, e184.[CrossRef][Medline]
Jansen, G., Thijssen, K. L., Werner, P., van der Horst, M., Hazendonk, E., and Plasterk, R. H. (1999). The complete family of genes encoding G proteins of Caenorhabditis elegans. Nat. Genet 21, 414–419.[CrossRef][Medline]
Kays, A. M., Rowley, P. S., Baasiri, R. A., and Borkovich, K. A. (2000). Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa. Mol. Cell. Biol 20, 7693–7705.
Kim, H., and Borkovich, K. A. (2004). A pheromone receptor gene, pre-1, is essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa. Mol. Microbiol 52, 1781–1798.[CrossRef][Medline]
Kraakman, L., Lemaire, K., Ma, P., Teunissen, A. W., Donaton, M. C., Van Dijck, P., Winderickx, J., de Winde, J. H., and Thevelein, J. M. (1999). A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose. Mol. Microbiol 32, 1002–1012.[CrossRef][Medline]
Kruger, J., Loubradou, G., Regenfelder, E., Hartmann, A., and Kahmann, R. (1998). Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis. Mol. Gen. Genet 260, 193–198.[CrossRef][Medline]
Kubler, E., Mosch, H. U., Rupp, S., and Lisanti, M. P. (1997). Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism. J. Biol. Chem 272, 20321–20323.
Kwon-Chung, K. J., Edman, J. C., and Wickes, B. L. (1992). Genetic association of mating types and virulence in Cryptococcus neoformans. Infect. Immun 60, 602–605.
Lawler, O. A., Miggin, S. M., and Kinsella, B. T. (2001). Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling. J. Biol. Chem 276, 33596–33607.
Lengeler, K. B., Davidson, R. C., D'Souza, C., Harashima, T., Shen, W. C., Wang, P., Pan, X., Waugh, M., and Heitman, J. (2000). Signal transduction cascades regulating fungal development and virulence. Microbiol. Mol. Biol. Rev 64, 746–785.
Lin, X., Huang, J. C., Mitchell, T. G., and Heitman, J. (2006). Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATalpha allele enhances filamentation. PLoS Genet 2, e187.[CrossRef][Medline]
Lin, X., Hull, C. M., and Heitman, J. (2005). Sexual reproduction between partners of the same mating type in Cryptococcus neoformans. Nature 434, 1017–1021.[CrossRef][Medline]
Loftus, B. J. et al. (2005). The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307, 1321–1324.
Lorenz, M. C., and Heitman, J. (1997). Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog. EMBO J 16, 7008–7018.[CrossRef][Medline]
Lorenz, M. C., Pan, X., Harashima, T., Cardenas, M. E., Xue, Y., Hirsch, J. P., and Heitman, J. (2000). The G protein-coupled receptor Gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Genetics 154, 609–622.
Maidan, M. M., De Rop, L., Serneels, J., Exler, S., Rupp, S., Tournu, H., Thevelein, J. M., and Van Dijck, P. (2005). The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans. Mol. Biol. Cell 16, 1971–1986.
McCudden, C. R., Hains, M. D., Kimple, R. J., Siderovski, D. P., and Willard, F. S. (2005). G-protein signaling: back to the future. Cell Mol. Life Sci 62, 551–577.[CrossRef][Medline]
Metodiev, M. V., Matheos, D., Rose, M. D., and Stone, D. E. (2002). Regulation of MAPK function by direct interaction with the mating-specific Galpha in yeast. Science 296, 1483–1486.
Moore, T. D., and Edman, J. C. (1993). The -mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol. Cell. Biol 13, 1962–1970.
Neptune, E. R., and Bourne, H. R. (1997). Receptors induce chemotaxis by releasing the subunit of Gi, not by activating Gq or Gs. Proc. Natl. Acad. Sci. USA 94, 14489–14494.
Neptune, E. R., Iiri, T., and Bourne, H. R. (1999). Gi is not required for chemotaxis mediated by Gi-coupled receptors. J. Biol. Chem 274, 2824–2828.
Nielsen, K., Cox, G. M., Wang, P., Toffaletti, D. L., Perfect, J. R., and Heitman, J. (2003). Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates. Infect. Immun 71, 4831–4841.
Nomoto, S., Nakayama, N., Arai, K., and Matsumoto, K. (1990). Regulation of the yeast pheromone response pathway by G protein subunits. EMBO J 9, 691–696.[Medline]
Palmer, D. A., Thompson, J. K., Li, L., Prat, A., and Wang, P. (2006). Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans. J. Biol. Chem 281, 32596–32605.
Perfect, J. R., Ketabchi, N., Cox, G. M., Ingram, C. W., and Beiser, C. L. (1993). Karyotyping of Cryptococcus neoformans as an epidemiological tool. J. Clin. Microbiol 31, 3305–3309.
Regenfelder, E., Spellig, T., Hartmann, A., Lauenstein, S., Bolker, M., and Kahmann, R. (1997). G proteins in Ustilago maydis: transmission of multiple signals? EMBO J 16, 1934–1942.[CrossRef][Medline]
Shen, W. C., Davidson, R. C., Cox, G. M., and Heitman, J. (2002). Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans. Eukaryot. Cell 1, 366–377.
Shimizu, K., and Keller, N. P. (2001). Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 157, 591–600.
Simon, M. I., Strathmann, M. P., and Gautam, N. (1991). Diversity of G proteins in signal transduction. Science 252, 802–808.
Slessareva, J. E., Routt, S. M., Temple, B., Bankaitis, V. A., and Dohlman, H. G. (2006). Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome. Cell 126, 191–203.[CrossRef][Medline]
Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. (1998). A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. USA 95, 5187–5192.
Wang, P., Cutler, J., King, J., and Palmer, D. (2004). Mutation of the regulator of G protein signaling Crg1 increases virulence in Cryptococcus neoformans. Eukaryot. Cell 3, 1028–1035.
Wang, P., Nichols, C. B., Lengeler, K. B., Cardenas, M. E., Cox, G. M., Perfect, J. R., and Heitman, J. (2002). Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans. Eukaryot. Cell 1, 257–272.
Wang, P., Perfect, J. R., and Heitman, J. (2000). The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans. Mol. Cell. Biol 20, 352–362.
Wu, L., Valkema, R., Van Haastert, P. J., and Devreotes, P. N. (1995). The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium. J. Cell Biol 129, 1667–1675.
Xu, J., Wang, F., Van Keymeulen, A., Herzmark, P., Straight, A., Kelly, K., Takuwa, Y., Sugimoto, N., Mitchison, T., and Bourne, H. R. (2003). Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114, 201–214.[CrossRef][Medline]
Xue, C., Bahn, Y. S., Cox, G. M., and Heitman, J. (2006). G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans. Mol. Biol. Cell 17, 667–679.
Yu, J. H. (2006). Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans. J. Microbiol 44, 145–154.[Medline]
Zuber, S., Hynes, M. J., and Andrianopoulos, A. (2002). G-protein signaling mediates asexual development at 25°C but has no effect on yeast-like growth at 37°C in the dimorphic fungus Penicillium marneffei. Eukaryot Cell 1, 440–447.
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fused clamps; 
, unfused clamps (third and fourth rows of panels). The fusion efficiency in isogenic strain backgrounds was determined by quantitative cell–cell fusion assays (bottom row of panels).
