INTRODUCTION

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Fungi are eukaryotic organisms that diverged from a common ancestor with multicellular eukaryotic animals some 800 to 1,000 million years ago. Despite this evolutionary divergence, fungi are more closely related to animals than to plants, algae, bacteria, or archea and thus share important features with mammalian cells. This is perhaps most apparent in the signaling cascades that regulate cell function. The yeast Saccharomyces cerevisiae expresses at least three members of the G protein-coupled family of serpentine receptors, which are in turn coupled to a heterotrimeric G protein and a G protein alpha subunit homolog. Mating in yeast cells is regulated by a mitogen-activated protein (MAP) kinase cascade that is highly conserved with MAP kinase cascades in mammalian cells. Finally, the signal transduction components that are targeted by the immunosuppressive drugs cyclosporin A, FK506, and rapamycin are remarkably conserved from yeasts to humans. Thus, studies on signal transduction in S. cerevisiae and other genetically tractable fungi promise to reveal common conserved mechanisms of signal transduction.

Recent studies have revealed that the model yeast S. cerevisiae undergoes a dimorphic transition to filamentous growth in response to nutritional signals in the environment, particularly nitrogen limitation. Filamentous growth occurs in both haploid and diploid cells in different environments and may play novel roles in the life cycle of this organism. At least two conserved signal transduction cascades that regulate filamentous growth have been defined, and remarkably related signaling pathways also operate during differentiation of other fungi, including pathogens of both plants and animals. These recent findings suggest that S. cerevisiae is also an excellent model system with great potential to provide insights into signaling in other fungi. We review here studies on the signal transduction cascades that regulate yeast filamentous growth and the related signaling pathways that operate in the fission yeast Schizosaccharomyces pombe, in human fungal pathogens (Candida albicans and Cryptococcus neoformans), in plant fungal pathogens (Ustilago maydis, Magnaporthe grisea, and Cryphonectria parasitica), and in model filamentous fungi (Aspergillus nidulans and Neurospora crassa). Taken together, these studies reveal a high degree of conservation between divergent organisms and illustrate conserved basic principles in the molecular determinants of life. Several other excellent reviews on yeast signal transduction have been published recently and also cover some additional topics in fungal development (5, 8, 10, 17, 18, 30, 139, 140, 177, 178, 263, 276, 289, 290).

In response to nitrogen limitation and abundant fermentable carbon source, diploid cells of S. cerevisiae undergo dimorphic transition to a filamentous growth form referred to as pseudohyphal differentiation (93) (Fig. 1 and 2). Filamentous growth represents a dramatic change in the normal pattern of cell growth in which the cells become elongated, switch to a unipolar budding pattern, remain physically attached to each other, and invade the growth substrate (Fig. 2). This alternative growth form may enable this nonmotile species to forage for nutrients under adverse conditions.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Life cycle of S. cerevisiae. Haploid yeast cells mate on rich medium. Diploid yeast cells adopt alternative fates depending on the availability of nutrients. N + C, abundant nitrogen and fermentable carbon source; n + C, limiting nitrogen and abundent fermentable carbon source; n + c, limiting nitrogen and nonfermentable carbon source; , no nutrients.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Filamentous and invasive growth of S. cerevisiae. On the left, invasive growth of haploid and diploid wild-type (wt) and tpk2 mutant yeast colonies is shown. On the right, the typical morphology of yeast pseudohyphal colonies is depicted, and a tpk2/tpk2 mutant with a filamentous growth defect is shown for comparison.

At least two conserved signaling pathways regulate yeast filamentous growth (Fig. 3). The first cascade involves components of the MAP kinase pathway that is also required for mating in haploid yeast cells in response to pheromones (56, 161, 180). The components of this MAP kinase cascade required for filamentous growth include the Ste20, Ste11, Ste7, and Kss1 kinases and the Ste12 transcription factor. In addition, another transcription factor, Tec1, forms a heterodimer with Ste12 that regulates expression of Tec1 itself and additional targets, such as the cell surface flocculin Flo11 required for agar invasion and filamentation (90, 165, 176). The pheromones, pheromone receptors, and subunits of the pheromone-activated heterotrimeric G protein are dispensable for filamentous growth and are not expressed in diploid cells (161).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Signal transduction cascades regulating pseudohyphal differentiation in S. cerevisae. Two parallel signaling cascades regulating filamentous growth are depicted: a nutrient-sensing cAMP-PKA pathway, and the MAP kinase cascade. AC, adenylylcyclase.

Thus, S. cerevisiae is able to use common components and yet couple them into two different pathways that sense two different environmental signals and give rise to two completely different developmental fates: mating in haploid cells in response to pheromone, and filamentous growth in diploid cells in response to nitrogen limitation and other environmental signals (Fig. 1). Signaling specificity is achieved by at least four different specializations in this signaling pathway (Fig. 4). First, the MAP kinase cascade is activated by the subunits of the pheromone-activated heterotrimeric G protein during mating of haploid cells. During filamentous growth, the MAP kinase pathway is activated by a different mechanism involving the Cdc42, Ras2, and 14-3-3 proteins Bmh1 and Bmh2 (201, 203, 236). Second, the components of the MAP kinase cascade are tethered together by the scaffold protein Ste5 during mating, whereas Ste5 is not required during filamentous growth, and another protein may serve this scaffolding function. It has been suggested that the Spa2 protein, which physically interacts with many of the MAP kinase cascade components, might function as the scaffold during filamentous growth (239). The third level of specialization is at the level of the MAP kinase itself. In S. cerevisiae, the Fus3 and Kss1 kinases have diverged, so that Fus3 is specialized to regulate mating and actually inhibits invasive growth, whereas Kss1 is specialized to regulate invasive and filamentous growth (56, 180). In addition, Kss1 has both positive and negative regulatory roles during filamentous growth (21, 22, 176, 178), in part by relieving repression of Ste12 by the Dig1 and Dig2 proteins (55). Finally, during mating Ste12 interacts with the Mcm1 protein to activate transcription of genes containing pheromone response elements, whereas in diploid cells Ste12 forms a heterodimer with Tec1 that activates transcription of genes with filamentation response elements (176). In this way, one common protein, Ste12, can yield two different patterns of appropriate transcriptional responses in haploid and diploid cells.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   Signaling specificity during mating and pseudohyphal growth in S. cerevisiae. Two related MAP kinase signaling cascades allow haploid and diploid yeast cells to respond to two different environmental signs, pheromone and nitrogen limitation, and give rise to two completely different developmental fates, mating in haploid cells and filamentous growth in diploid cells. Signaling specificity is achieved by at least four mechanisms. First, pheromone activates a G protein whose subunits activate the MAP kinase cascade, and these components are not expressed in diploid yeast cells and do not regulate filamentous growth. Second, the Ste5 scaffold tethers the components during mating but does not play a role in diploid filamentous growth. Third, the MAP kinase has diverged and specialized: Fus3 to regulate mating and Kss1 to regulate filamentous growth. Finally, Ste12 homodimers or heterodimers with Mcm1 activate pheromone response element-regulated genes in mating or with Tec1 to regulate filamentation response element-driven genes during diploid filamentous growth.

A second signaling pathway functions in parallel with the MAP kinase pathway to regulate pseudohyphal differentiation (Fig. 3). This second pathway is a nutrient-sensing pathway and involves a novel G protein-coupled receptor, Gprl, the G proteins Gpa2 and Ras2, adenylyl cyclase, cyclic AMP (cAMP), and cAMP-dependent protein kinase (12, 143, 171, 172, 223, 238, 276, 314). In S. cerevisiae, three genes encode the catalytic subunits of cAMP-dependent protein kinase, which play redundant roles in vegetative growth but specialized roles in filamentous growth. The Tpk2 catalytic subunit positively regulates filamentous growth by regulating the transcription factor Flo8, which in turn regulates Flo11 expression (223). Flo11 is a glycosyl-phosphatidylinositol (GPI)-linked cell surface protein that is required for pseudohyphal and haploid invasive growth. In particular, Flo11 plays a role in mother-daughter cell adhesion, which is required for the integrity of pseudohyphal filaments (150, 165). Flo8 was previously shown to be required for pseudohyphal differentiation, and the common lab strain S288C harbors a naturally occurring flo8 mutation that prevents filamentous differentiation (162). Tpk2 also inhibits a transcriptional repressor, Sfl1, which also regulates Flo11 expression (238). The Tpk1 and Tpk3 catalytic subunits play a negative role in regulating filamentous growth, possibly by a feedback loop that inhibits cAMP production (212).

Yeast cells express only two heterotrimeric G protein subunits: Gpa1, which plays a well-established role in mating, and Gpa2, which was discovered in 1988 by low-stringency hybridization with a mammalian G subunit but whose physiological function was unknown for many years (207). Gpa2 was subsequently discovered to be required for yeast filamentous growth, but it signals in a pathway distinct from the MAP kinase cascade (143, 171). Filamentous growth of gpa2 mutant strains is restored by exogenous cAMP, suggesting that Gpa2 regulates a cAMP signaling pathway regulating filamentous growth. In fact, earlier studies had suggested that Gpa2 might play a role in regulating cAMP production in response to extracellular glucose (207).

The Gpr1 G protein-coupled receptor was subsequently identified by a two-hybrid screen with the G protein Gpa2 (314, 322). This is one of only a few examples in which integral membrane proteins have been studied in the two-hybrid system; in this case, the C-terminal soluble tail of Gpr1 was found to interact with the coupled G protein Gpa2. The Gpr1 receptor was found to play a role important for vegetative growth, and gpr1 mutations are nearly synthetically lethal with ras2 mutations (314). Recent evidence suggests that the ligand of the Gpr1 receptor may be glucose and other structurally related fermentable sugars. When yeast cells are starved for glucose, readdition of glucose triggers a rapid and transient increase in intracellular cAMP levels (reviewed in reference 276). Most interestingly, both the Gpr1 receptor and the G protein Gpa2 are required for cAMP production in response to glucose readdition (54, 137, 172, 321). The Gpr1 receptor is coupled to the heterotrimeric G protein  subunit Gpa2 and is also required for pseudohyphal differentiation and plays a role in nutrient sensing (12, 172, 223, 271). Early studies suggested Gpa2 might stimulate cAMP production by adenylyl cyclase. Consistent with this, cAMP stimulates pseudohyphal differentiation and suppresses the filamentation defects of both gpr1 and gpa2 mutant strains (143, 171, 172, 223). Dominant activated Gpa2 mutations also suppress the pseudohyphal defect of mutant strains lacking the Gpr1 receptor, supporting the hypothesis that Gpa2 signals downstream of Gpr1.

Interestingly, several observations suggest that the Gpr1-Gpa2 receptor system plays a dual role in sensing both fermentable carbon sources and limiting nitrogen source. First, expression of the GPR1 receptor gene is dramatically induced by nitrogen starvation (314). Second, cAMP or dominant active alleles of Gpa2 signal filamentous growth even when nitrogen levels are increased to levels that repress differentiation of wild-type strains (171). Third, Gpa2 has been shown to bind to and inhibit the meiotic regulatory kinase Ime2 specifically under nitrogen-limiting conditions (74). These findings suggest that the sensitivity of this carbon-sensing mechanism is enhanced under nitrogen starvation conditions and that Gpa2 may receive input from other sources that sense nitrogen levels.

Interestingly, a recent report has suggested that the yeast phospholipase C homolog is in a physical complex with the Gpr1 receptor and is required for association of the G subunit Gpa2 with the receptor and for pseudohyphal differentiation (12). Plc1 cleaves PIP₂ to produce inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) (83). Recent studies have revealed two inositol kinases, Ipk1 and Ipk2, which phosphorylate IP₃ and its products (245, 246, 318). IP₃ is phosphorylated to IP₄ and IP₅ by the dual-specificity kinase Ipk2, which regulates transcription (219), and the Ipk1 kinase then phosphorylates IP₅ to produce IP₆, which regulates mRNA export from the nucleus (318). Some studies have suggested that glucose regulates a pathway involving Ras and Plc1 in phosphatidylinositol metabolism and regulation of the plasma membrane H⁺-ATPase (32, 53, 124). Further studies will be required to eludicate the role of Plc1 in yeast filamentous growth. In summary, a second signaling pathway comprising the Gpr1 receptor, the Gpa2 G protein, and cAMP regulates pseudohyphal growth in parallel to and independently of the MAP kinase pathway (Figure 3).

The target of cAMP in S. cerevisiae is the cAMP-dependent protein kinase PKA. Yeast and mammalian PKA are similar, and both enzymes consist of a regulatory and a catalytic subunit. In yeast cells, the PKA regulatory subunit is encoded by a single gene, BCY1, and three catalytic subunits are encoded by the TPK1, TPK2 and TPK3 genes (39, 279, 280). In both S. cerevisiae and mammals, PKA in resting cells is an inactive tetramer composed of two regulatory subunits bound to two catalytic subunits. In response to external signals that increase intracellular cAMP levels, cAMP binds to the regulatory subunit and triggers conformational changes that release the active catalytic subunits. The yeast cAMP-dependent protein kinase is required for vegetative growth. Triple mutants lacking the Tpk1, Tpk2, and Tpk3 catalytic subunits are inviable, whereas mutant strains expressing any one of the three Tpk subunits are viable. These findings led to the model that the three PKA catalytic subunits are largely redundant for function.

Recent studies reveal that cAMP-dependent protein kinase plays a central role in regulating yeast pseudohyphal differentiation (223, 238). First, mutations of the PKA regulatory subunit Bcy1 dramatically enhance filamentous growth (223). Second, the PKA catalytic subunits play distinct roles in regulating filamentous growth: the Tpk2 subunit activates filamentous growth (Fig. 2), whereas the Tpk1 and Tpk3 subunits primarily inhibit filamentous growth (223, 238). The unique activating function of the Tpk2 subunit is linked to structural differences in the catalytic region of the kinase and not to differences in gene regulation or the unique amino-terminal region of the protein (223). Genetic epistasis experiments support a model in which Tpk2 functions downstream of the Gpr1 receptor and the G protein Gpa2 (172, 223). Importantly, activation of PKA by mutation of the Bcy1 regulatory subunit restores pseudohyphal growth in mutants lacking elements of the MAP kinase pathway, including ste12, tec1, and ste12 tec1 mutants (223, 243). Thus, the MAP kinase and PKA pathways independently regulate filamentous growth. Further analysis reveals that the PKA pathway regulates the switch to unipolar budding and invasion, whereas the MAP kinase pathway is required for cell elongation and invasion (Fig. 3).

Recent studies have defined a role for the PKA pathway in activating pseudohyphal growth via transcriptional regulation of the cell surface flocculin Flo11 by the Flo8 transcription factor (223, 243). The transcriptional repressor Sfl1 was also found to interact with the Tpk2 catalytic subunit in a two-hybrid screen (238). Tpk2 appears to enhance Flo11 expression by inhibiting the repression activity of Sfl1. How PKA regulates either Flo8 or Sfl1 is not yet understood in molecular detail. The FLO11 gene promoter is extremely large, ~3,000 bp, and is regulated by a complex set of transcription factors that includes Ste12/Tec1, Flo8, Sfl1, Msn1/Mss10/Phd2, and Mss11 (87, 243). Taken together, these findings reveal an intimate role for the cAMP-dependent kinase in the regulation of yeast dimorphism.

Two other proteins that appear to regulate the Gpr1-Gpa2-cAMP signaling pathway under certain physiological conditions have recently been identified. One is the low-affinity cAMP phosphodiesterase Pde1 (175). Yeast cells express two different cAMP phosphodiesterases: a high-affinity form called Pde2, and a low-affinity form called Pde1. Interestingly, pde1 mutations dramatically enhance production of cAMP in response to glucose readdition, whereas pde2 mutations have little effect (175). This is in contrast to pseudohyphal differentiation, where pde2 mutations enhance filamentous growth (143, 171, 223). The effects of pde1 mutations on filamentous growth are not yet known. The Pde1 enzyme has a single PKA consensus phosphorylation site and is a phosphoprotein in vivo, and phosphorylation in crude extracts leads to modest increases in enzyme activity (175). These findings suggest that Pde1 could be part of the feedback loop that limits cAMP excursions in response to glucose signaling. A second novel regulator of the pathway is a regulator of G protein signaling (RGS) protein homolog, Rgs2, which binds to the Gpa2-GTP complex and stimulates GTP hydrolysis (288). rgs2 mutations enhance cAMP production in response to glucose, whereas overexpression of Rgs2 attenuates this response. In this regard, Rgs2 functions analogously to the RGS protein Sst2, which stimulates GTP hydrolysis of the Gpa1-GTP complex to attenuate the pheromone response in haploid yeast cells. A role for Rgs2 in pseudohyphal differentiation has not yet been established.

In summary, studies on pseudohyphal differentiation have resulted in the elucidation of several novel signaling features of the PKA pathway. First is the identification of a novel G protein-coupled receptor, Gpr1, which may be the first known G protein-coupled receptor whose ligand is a nutrient. Interestingly, Gpr1 is conserved in C. albicans and S. pombe, and the coupled G protein is known to be present in many different fungi. Second, the G protein Gpa2 has sequence identity with other G subunits of heterotrimeric G proteins, and it appears to be coupled to a classic G protein-coupled receptor. However, no associated subunits have been identified. A number of candidate  and  subunits have been mutated, but none conferred phenotypes related to pseudohyphal growth (171). We propose that Gpa2 may function either as a solo G subunit or in complex with other proteins such as Plc1 and Ras2. In either case, this represents a new paradigm for signaling from a G protein-coupled receptor via a very unusual type of G protein. Finally, a very interesting finding is the specialized role of the PKA catalytic subunits in regulating filamentous growth. The three catalytic subunits of PKA were thought to be functionally redundant because the essential vegetative function can be satisfied by expression of any one of the three. In contrast, Tpk2 plays a specialized role to activate filamentous growth, whereas Tpk1 and Tpk3 play an inhibitory role. The positive function of Tpk2 involves regulation of the Flo8 and Sfl1 transcription factors. The negative role of Tpk1 and Tpk3 may involve the known role of PKA in a feedback loop that inhibits cAMP production (212). Studies to localize the catalytic and regulatory subunits of PKA under different nutritional conditions have just begun (98) and may provide insights into the unique and specialized roles of Tpk2 compared to Tpk1 and Tpk3.

Recent studies have revealed several examples of cross talk between the MAP kinase and cAMP signaling pathways in the regulation of filamentous growth. First, the small G protein Ras2 plays a dual signaling role, activating both the MAP kinase signaling pathway and adenylyl cyclase (171, 201-203). Second, both the MAP kinase and the PKA signaling pathways converge to regulate the large, complex promoter of the FLO11 gene (223, 243), which encodes a cell surface protein required for pseudohyphal growth (150, 165). Third, cAMP inhibits the expression of MAP kinase-regulated reporter genes (171), and high levels of cAMP or PKA activity enhance filamentous growth but give rise to filaments comprised of round rather than elongated cells (223). Because the MAP kinase pathway regulates cell elongation, these observations suggest that the PKA pathway may antagonize signaling by the MAP kinase pathway at some level. Recent findings have suggested that during haploid invasive growth, PKA may also positively regulate signaling by the MAP kinase cascade at a level downstream of the Ste20 kinase (202). The Ste12 transcription factor has several consensus PKA phosphorylation sites, and overexpression of Ste12 can in some cases suppress pseudohyphal defects, whereas the constitutive active Ste11-4 mutant does not (170), suggesting that Ste12 could represent a target of the PKA pathway that is both positively and negatively regulated depending on cell type.

Pseudohyphal growth occurs in diploid cells in response to the presence of an abundant fermentable carbon source and limiting nitrogen source. A related morphological process, termed haploid invasive growth, has also been described, which shares some features with diploid pseudohyphal growth (237) (see Fig. 2). During diploid filamentous growth, some cells invade the agar to produce chains of cells that cannot be removed by vigorous washing. Although haploid strains do not undergo pseudohyphal differentiation under standard conditions, haploid strains derived from the 1278b background can invade the agar when grown on rich medium for an extended period of time. Many of the same signaling components that regulate diploid filamentous growth are also required for haploid invasive growth, including several components of the MAP kinase pathway (Ste20, Ste11, Ste7, Ste12, and Tec1) (237). The two related MAP kinases play opposing roles, and fus3 mutants are hyperinvasive whereas kss1 mutants have a defect in agar invasion. Components of the PKA pathway also regulate filamentous growth, including Gpr1, Gpa2, Ras2, Tpk2, Flo8, and Flo11 (165, 172, 202, 223). Taken together, these findings suggest that haploid invasive growth shares many features with diploid filamentous growth.

In contrast to diploid filamentous growth, which occurs on nitrogen-limiting medium, haploid invasive growth normally occurs on rich growth media, including YPD (yeast extract-peptone-dextrose) medium. It has been suggested that nutritional limitation might occur beneath colonies and stimulate haploid invasive growth, even on rich medium. When yeast cells begin to exhaust nutrients in rich medium, cellular proteins and amino acids are catabolized to produce nitrogen, resulting in the production of short-chain alcohols derived from amino acids, which are called fusel oils. Recent studies reveal that several short-chain alcohols, including isoamyl alcohol and butanol, dramatically stimulate pseudohyphal differentiation of haploid yeast strains (72, 168). How these alcohols are sensed is not yet known, but these or other metabolic products could regulate differentiation under certain culture conditions.

Other recent findings reveal that mating pheromones regulate invasive and filamentous growth of haploid S. cerevisiae strains (see Fig. 11). In the wild, most strains of S. cerevisiae are diploid, and the haploid state of the life cycle essentially represents gametes that are short-lived in nature. An elaborate pattern of axial budding has evolved in haploid yeast cells and is thought to promote more rapid mating and diploidization following meiosis. Thus, the main activity of haploid yeast strains would appear to be locating a mating partner. Recent studies using genome arrays reveal that pheromone induces several genes that are known to be induced during filamentous growth, including a hydrolytic enzyme encoded by the PGU1 gene (179, 235).

Remarkably, low concentrations of mating pheromones were found to increase agar-invasive growth (235) (see also Fig. 11). These observations suggest that haploid invasive growth may be a mechanism by which yeast cells can locate mating partners at a distance, and yeast cells are known to be responsive to gradients of mating pheromone. During invasive growth, the haploid cells become elongated, switch from an axial pattern of budding to bipolar and unipolar budding, and invade the agar (235, 237). Thus, while the role of filamentous growth in diploids may be to forage for nutrients, the role in haploid cells may be to forage for mating partners. Haploid invasive growth can occur to some extent in the absence of a mating partner. This may be attributable to a basal level of signaling in the absence of ligand. Alternatively, low concentrations of pheromone may result from cells that have switched mating type in the culture or in which repression of the silent mating type cassettes is inefficient, as is known to be the case in older yeast cells. The finding that the pheromone receptors and coupled G protein are not required for standard haploid invasive growth indicates that pheromones may not normally be involved. Pheromone-induced invasive growth requires Ste12 but is independent of Tec1 (235), whereas haploid invasive growth in the absence of pheromone requires both Ste12 and Tec1 (237). Interestingly, filamentous differentiation of haploid S. cerevisiae cells that occurs in response to mating pheromones is analogous to the recent discovery that filamentation and sporulation (haploid fruiting) of MAT cells of the human fungal pathogen C. neoformans are dramatically induced by factors secreted by MATa mating partner cells (293) (see Fig. 11). Taken together, these studies illustrate that mating and filamentous growth are linked, which is perhaps most apparent in the basidiomycetes in which mating results in a filamentous dikaryon.

These observations on the activation of filamentous growth by pheromone may be related to the previous finding that filamentation reporter genes are inappropriately activated by basal signaling of the pheromone response pathway in fus3 mutant haploid cells (177). This example of cross talk requires the pheromone-activated G subunit Ste4, occurs via misactivation of Kss1 on the Ste5 scaffold, and can be further enhanced by mating pheromone.

Another example of cross talk between the MAP kinase and cAMP signaling pathways occurs in haploid cells responding to pheromone. Normally, when glucose is readded to yeast cells that have been starved for glucose, cAMP levels increase (reviewed in reference 276). In contrast, in haploid cells that have first been exposed to mating pheromone, readdition of glucose fails to stimulate cAMP production (13). The pheromone receptor Ste2 and the  subunit of the heterotrimeric G protein (Ste4) are required for inhibition of cAMP production by pheromone, but further-downstream components of the pheromone signaling pathway are not. Expression of a constitutively activated Ras2 mutant (Val-19) restored cAMP production in the presence of pheromone, and pheromone failed to inhibit the modest increase in cAMP in response to glucose that still occurs in a gpa2 mutant strain (225). Taken together, these findings support a model in which pheromone-stimulated release of the subunit of the heterotrimeric G protein inhibits activation of adenylyl cyclase by the G subunit Gpa2. Although Gpa2 does not appear to have its own dedicated subunit, this cross talk between the pheromone-regulated G protein and Gpa2 could serve to inhibit signaling via the PKA pathway under certain conditions, such as high levels of pheromone, to promote mating and inhibit filamentous growth. This pathway can only operate in haploid cells, and not during diploid filamentous growth, because the pheromones, pheromone receptors, and subunits are only expressed in haploid cells.

Other proteins that regulate yeast filamentous growth have been identified that do not appear to be components of either the MAP kinase or the cAMP signaling pathway. These include the ammonium transporter Mep2, which is required for filamentous differentiation in response to ammonium-limiting conditions (169), and the transcription factors Phd1, Sok2, and Ash1 (43, 92, 224, 296). How limiting nitrogen sources are sensed during pseudohyphal differentiation is not understood in detail. Map 2 ammonium permease is required for filamentous differentiation and plays a unique role not shared with the related Mep1 and Mep3 ammonium permeases (169, 170). mep/mep2 mutant strains fail to differentiate but have no growth defect on medium limiting for ammonium, leading to the hypothesis that the Mep2 protein may function to both transport and sense ammonium ions (169). The Gln3 transcriptional activator and the Gln3 repressor Ure2 that regulate the nitrogen catabolite response (NCR) are also required for pseudohyphal growth. Both gln3 mutants unable to induce the NCR response and ure2 mutants in which the NCR response is constitutive fail to differentiate, suggesting that the ability both to induce and to repress the NCR genes involved in nitrogen source utilization is required for filamentous differentiation (169). The GATA family transcription factor Ash1 represses HO endonuclease expression in haploid daughter cells, restricting mating type switching to haploid mother cells. Ash1 is also required for pseudohyphal differentiation and is localized to daughter cell nuclei in filament cells (43), where it may function to regulate Flo11 expression (224). The transcription factor Sok2 was recently found to regulate a transcription factor cascade involving Phd1, Swi5, and Ash1, which in turn regulates expression of proteins and enzymes (Flo11, Egt2, and Cts1) involved in mother-daughter cell adhesion and separation (224). Several additional proteins also appear to inhibit filamentous growth, including the Elm1 protein kinase homolog and the B subunit of protein phosphatase 2A (Cdc55) (28). Recent studies suggest that these proteins regulate Cdc28 via the Hsl1-Hsl7-Swe1 regulatory cascade and that the Cdc28-cyclin complexes regulate filamentous growth (76).

In addition, several recent studies indicate that both the G₁ cyclins Cln1, Cln2, and Cln3 and the Clb2 mitotic cyclin may play roles in filamentous growth (6, 167). cln1 and cln2 mutations inhibit filamentous growth, whereas cln3 mutations enhance filamentation. Mutations in the F-box component of the Skp1-Cdc53-F box protein (SCF) ubiquitin ligase complex, Grr1, dramatically enhance the stability of Cln1 and Cln2 and enhance filamentous growth. Epistasis analysis suggests that Cdc28-Cln1/2 may act at an early step in the MAP kinase cascade (167). In addition, the Cdc28-Cln1/2 complex can phosphorylate Ste20, and cln1 and cln2 mutations are synthetically lethal with mutations in the CLA4 gene, which encodes an Ste20 homolog (220, 307). These findings imply that the MAP kinase cascade may be regulated at the level of Ste20 by Cdc28 in complex with G₁ cyclins. Further studies will be required to understand these additional regulatory elements in molecular detail.

Like budding yeast, the fission yeast S. pombe is homothallic and has a defined sexual cycle involving mating between haploid cells of opposite mating types; in S. pombe, the mating partners are called h⁺ and h cells (reviewed in reference 315). In contrast to S. cerevisiae, mating in S. pombe is regulated by both pheromones and nutrients. Nitrogen limitation induces many of the components of the pheromone response pathway and is required for mating (reviewed in references 78 and 211). Also in contrast to S. cerevisiae, the diploid state of S. pombe is relatively unstable, and meiosis and sporulation usually occur immediately following cell fusion during mating. The difference in life style between these two yeasts is thought to have arisen because S. cerevisiae has evolved primarily to be diploid in the wild, whereas S. pombe occurs predominantly as a haploid in nature. As a consequence, the diploid yeast S. cerevisiae responds to nutrient limitation by undergoing either pseudohyphal differentiation or meiosis and sporulation, whereas when the haploid yeast S. pombe encounters adverse nutritional conditions, it must first mate and can then undergo meiosis and sporulate.

This difference in life cycle has led to an interesting difference in the signaling pathways regulating mating in the two yeasts. In S. cerevisiae, mating is primarily regulated by a linear signaling pathway composed of the pheromone-activated MAP kinase cascade. In S. pombe, two signaling pathways coordinately regulate mating (Fig. 5). One is a pheromone-activated MAP kinase signaling pathway similar to the one that operates during mating in S. cerevisiae. The second is a nutrient-regulated, G protein-cAMP-PKA signaling pathway quite similar to the nutrient-sensing Gpr1-Gpa2-PKA pathway that regulates pseudohyphal differentiation in S. cerevisiae. Thus, in one organism (S. cerevisiae), these two signaling pathways have evolved cell type specificity, so that one functions in response to pheromone during mating and both function to detect nutrients in diploid cells for filamentous growth (Fig. 3 and 6). In contrast, in the other organism (S. pombe), these two pathways function coordinately in the same cell type to regulate mating under adverse nutrient conditions and in response to pheromone (Fig. 5).

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Signaling pathways regulating mating of S. pombe. Similar to S. cerevisiae diploid pseudohyphal growth, mating in S. pombe is regulated by two parallel signaling pathways. One responds to pheromones and involves a conserved MAP kinase (MAPK) signaling pathway. The second is a nutrient-sensing pathway that, in the presence of abundant nutrients, stimulates cAMP production to repress ste11 expression and inhibit mating. CAP, cyclase-associated protein.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Role of G proteins in pheromone response in budding and fission yeasts and the human fungal pathogen C. neoformans. The G protein subunits regulating pheromone and nutrient responses in these organisms are depicted. Dashed arrows depict weaker signaling.

A conserved nutrient-sensing signaling pathway has been defined that regulates mating and gene repression in S. pombe (Fig. 5). The components of this pathway were identified by two different approaches. In the first, Yamamoto and colleagues defined a role for cAMP in repressing mating and then set out to identify components of the cAMP signaling pathway (195, 315). In the second, Hoffman and Winston discovered that glucose dramatically repressed expression of the fbp1 gene, encoding fructose-1, 6-bisphosphatase, and by using lacZ reporter fusions isolated mutations in 10 complementation groups that block repression by glucose to identify the git genes (glucose-insensitive transcription) (38, 109, 110).

By these two different approaches, the elements of a conserved signaling pathway that senses nutrients and regulates cAMP production were defined. This pathway is remarkably similar to the Gpr1-Gpa2-cAMP-PKA pathway discussed above that regulates pseudohyphal differentiation in S. cerevisiae. As in budding yeast, glucose stimulates cAMP production in S. pombe, and classic studies revealed that either glucose limitation or nitrogen limitation results in a reduction in cAMP levels in S. pombe (195). cAMP levels decline rapidly in response to glucose starvation, whereas cAMP levels decline more gradually in response to nitrogen limitation, suggesting that there are different levels of control for carbon compared to nitrogen source sensing. S. pombe expresses git3, which is a homolog of the S. cerevisiae Gpr1 receptor (298). Interestingly, git3 lacks the asparagine-rich third intracellular loop found in Gpr1, indicating that this unusual region does not play an obligate role in signaling. The git3 G protein-coupled receptor homolog is required for glucose-induced repression of the fbp1 gene, and git3 mutants mate on nutrient-rich medium (38, 298). Genetic studies support a model in which the git3 receptor is coupled to the G subunit gpa2 (118, 213, 298). gpa2 is most homologous to the nutrient-sensing Gpa2 protein of S. cerevisiae that is required for pseudohyphal growth (143, 171). In contrast to wild-type cells, gpa2 mutant cells mate under nutrient-rich conditions, indicating a defect in nutrient sensing.

Most interestingly, the single G subunit that has been identified in S. pombe is required for adenylyl cyclase activation in response to glucose and repression of the fbp1 gene by glucose (151). Moreover, the phenotype of gpb1 mutant cells is identical to that of gpa2 mutant strains, namely, mating under nutrient-rich conditions. These findings suggest that gpb1/git5 is coupled to the gpa2 G protein and not to the pheromone-activated gpa1 G protein, in contrast to an earlier report (130) (Fig. 7). gpb1/git5 is not required for basal cAMP production, whereas gpa2 is, suggesting some G-independent action of gpa2 on adenylyl cyclase.

View larger version (11K): [in this window] [in a new window]   FIG. 7.   Role of G proteins in filamentous growth and mating in budding and fission yeasts. In both yeasts, two G proteins regulate cellular responses to pheromone and nutrients. In both, one functions as a heterotrimeric G protein with a partner, and the other signals in conjunction with Ras. Interestingly, the heterotrimeric G protein is coupled to pheromone receptors in S. cerevisiae but to the nutrient sensor in S. pombe. Importantly, the subunits appear to function with only one of the two G subunits in both organisms. MAPK, MAP kinase; AC, adenylylcyclase.

Additional git mutations that render transcription of the fbp1 gene insensitive to glucose are suppressed by cAMP and encode functions upstream of adenylyl cyclase (cyr1/git2), including git1, git7, and git10. These additional components may represent the cyclase-associated protein, the G subunit, hexokinase, or Plc1, all of which are known or would be predicted to regulate glucose signaling to adenylyl cyclase in other systems.

Several other components of the cAMP signaling pathway in S. pombe have also been identified. They include adenylyl cyclase itself, cyr1/git2. Quite interestingly, Hoffman and Winston discovered examples of intragenic complementation between different alleles of adenylyl cyclase (109). This unusual genetic behavior usually indicates that a protein has two functional domains. Consistent with this notion, alleles of one class of git2 mutations had no defect in cAMP production in vitro, whereas the other class failed to make cAMP in extracts. These findings suggest that adenylyl cyclase may exist as a dimer in which different domains of the protein have unique functions. In addition to adenylyl cyclase, both the regulatory (cgs1) and the catalytic subunits (pka1) of cAMP-dependent protein kinase have been identified (69, 182). PKA represses the transcription of both the ste11 gene (266), a key regulator of many mating-specific genes, and the fbp1 gene.

To summarize (see also Fig. 15), when glucose levels are high, cAMP is produced, PKA is active, and mating of S. pombe is repressed. When glucose or nitrogen levels fall, cAMP production is reduced, PKA is less active, and ste11 is induced. An important point is that because the role of PKA is to mediate repression of ste11, cAMP has a negative effect on mating in S. pombe. This is in contrast to the positive role that cAMP plays in promoting filamentous growth in S. cerevisiae. Because the pathways can be wired to have either positive or negative regulatory effects, one can essentially achieve either outcome with one common input to the signaling cascade.

By analogy with S. cerevisiae, the most likely ligand for the git3 receptor is glucose. An important unsolved question, then, is the role of nitrogen limitation in mating in S. pombe. Most interestingly, mating and meiosis occur in response to either nitrogen or glucose limitation. This suggests that nitrogen signaling also impinges on the git3-gpa2 signaling pathway or that overlapping signaling pathways sense glucose and nitrogen sources. One very intriguing recent report is that the addition of methionine can stimulate mating of S. pombe on nutrient-rich medium, which normally represses mating (249). One possible model is that methionine biosynthesis is stimulated and represses cAMP production in response to nitrogen source limitation. cAMP levels were found to decline to lower levels in cells cultured with methionine. However, methionine also restored ste11 and fbp1 expression in cells exposed to high levels of exogenous cAMP, suggesting that the effects of methionine may occur downstream or independently of cAMP production (249). We found no effect of methionine, cysteine, S-adenosylmethionine, S-adenosylhomocysteine, or biosynthetic intermediates of either amino acid on pseudohyphal growth in S. cerevisiae (M. E. Cardenas and J. Heitman, unpublished results), suggesting that this aspect of nitrogen sensing may differ in the two yeasts.

The components of a pheromone-regulated MAP kinase signaling pathway that coordinately regulates mating in S. pombe have also been identified (Fig. 5, 6, and 7). The P- and M-pheromones have been identified and synthesized, and the pheromone receptors (map3 and mam2) have also been identified, sequenced, and mutated (reviewed in reference 315). These components are required for mating in S. pombe and serve to regulate the production of conjugation tubes and early cell fusion steps. As discussed below, these components also function later and are required for meiosis of the diploid cell. The pheromone receptors are coupled to what appears to be the  subunit of a heterotrimeric G protein, gpa1 (215). Importantly, gpa1 plays an active role in mating (Fig. 6 and 7). gpa1 mutants are sterile, and dominant active alleles trigger conjugation tube formation in both mating types. The active signaling role played by the G subunit gpa1 is in contrast to budding yeast, in which the G subunit signals mating and the G subunit couples the G subunit to the receptor, inhibits signaling by G in the absence of pheromone, and also functions in adaptation. Only one G subunit has been identified, and its function is clearly coupled to the nutrient sensor and not to the pheromone receptor. This is similar to the situation in budding yeast, in which the G subunit Ste4 is coupled to only one of the two G subunits, in that case to the pheromone-regulated Gpa1 protein (Fig. 6). These observations suggest that, in contrast to mammals, in which G subunits are thought to interact with and regulate multiple G subunits, in fungi G subunits are coupled to only one of the two G subunits. This implies either that the other G subunit, gpa1 in S. pombe and Gpa2 in S. cerevisiae, functions with a nontraditional type of subunit, or that these G proteins function as solo G subunits, similar to other small GTP-binding proteins such as Ras.

Most interestingly, the ras1 homolog that has been identified in S. pombe functions in activating that MAP kinase pathway during mating and does not appear to play a prominent role in regulating cAMP production (85, 86, 206). In S. cerevisiae, Ras1 and Ras2 both regulate cAMP production by adenylyl cyclase, and Ras2 also regulates activation of the MAP kinase pathway during filamentous growth (202, 203). ras1 mutants of S. pombe have a mating defect, and dominant active ras1 alleles render cells hypersensitive to pheromone. The exchange factor that activates Ras1 to the GTP-bound form is called ste6, and ste6 expression is markedly induced by nitrogen starvation, suggesting that ras1 may be activated by regulated expression of its GDP-GTP exchange factor (115).

These studies illustrate a very interesting parallel with the signaling pathway regulating S. cerevisiae filamentous growth (Fig. 6 and 7). In that case, the nutrient-sensing G protein-coupled receptor (GPCR) Gpr1 is coupled to Gpa2, and Gpa2 in conjunction with Ras2 regulates cAMP production by adenylyl cyclase. There are no known G8 subunits for Gpa2. In S. pombe, it is instead the pheromone receptors that are coupled to a G subunit, gpa1, with no apparent G8 subunits, and again there is a similar role for signaling by both the G subunit gpa1 and the ras1 protein. We propose that these represent two examples of a novel class of GPCR-G protein system which lacks a classic heterotrimeric G protein. In these cases, the G subunit may be physically and functionally coupled to either or both Ras and phospholipase C and would thus represent very novel types of heterodimeric or heterotrimeric (but not ) G proteins. Clearly, further study of these very interesting signaling modules is of significant interest.

The components of a conserved MAP kinase module have also been found to regulate mating in S. pombe. These include homologs of the MAP kinase kinase kinase (byr2/ste8), the MAP kinase kinase (byr1/ste1), and a single MAP kinase (spk1). Interestingly, several of these components can function when heterologously expressed in S. cerevisiae mutant strains, suggesting that the pathway has been functionally conserved during the divergence of budding and fission yeasts (210).

Interestingly, mating pheromone signaling is also required for meiosis and sporulation of diploid S. pombe cells. This is in stark contrast to S. cerevisiae, in which, following cell fusion, the genes encoding pheromone receptors and pheromones are repressed and no longer expressed in the diploid zygote (263). This repression does not occur following fusion of haploid S. pombe cells, and continued pheromone signaling is required for meiosis and sporulation. Thus, diploid mutants lacking either the M-factor or the P-factor receptor undergo meiosis normally, but diploid mutants lacking both receptors are unable to undergo meiosis (272). Synthetic pheromones have also been employed to demonstrate regulation of meiosis-regulating genes by pheromone signaling (305).

These findings further underscore the intimate link that exists between mating, meiosis, and sporulation in fission yeast, which is similar to the life cycle of basidiomycetes, such as U. maydis and C. neoformans, in which pheromones regulate cell fusion and the heterokaryon then undergoes a dimorphic transition to filamentous growth. In the basidiomycetes, continued pheromone signaling is required for filament formation and may serve to signal clamp cell fusion during nuclear division and migration.

C. albicans is the most common human fungal pathogen and causes a wide range of superficial mucosal diseases as well as life-threatening systemic infections in immunocompromised patients (216). This fungus is dimorphic and can either grow as a budding yeast (blastospores) or switch to a filamentous form (either hyphae or pseudohyphae) under a variety of environmental conditions (216). The ability to undergo these reversible dimorphic switches is essential for the virulence of this pathogen (164). C. albicans is a diploid organism for which the sexual cycle has only very recently been discovered (117, 183). The characterization of a filamentous state in the baker's yeast S. cerevisiae has made it possible to use this as a model to compare regulation of filamentation in C. albicans and S. cerevisiae (93).

Multiple pathways have been shown to regulate the morphogenic transition between budding and filamentous growth in C. albicans (Fig. 8). A transcription factor, Cph1, which is homologous to the Ste12 transcription factor that regulates mating and pseudohyphal growth in S. cerevisiae, has been identified. (160, 184). In S. cerevisiae, Ste12 is regulated by a conserved MAP kinase cascade. A related MAP kinase cascade has been identified in C. albicans. The cascade consists of the kinases Cst20 (homologous to the p21-activated kinase [PAK] kinase Ste20), Hst7 (homologous to the MAP kinase kinase Ste7), and Cek1 (homologous to the Fus3 and Kss1 MAP kinases) (51, 132, 154, 299). Null mutations in any of the genes in the MAP kinase cascade (Cst20, Hst7, or Cek1) or the transcription factor Cph1 confer a hyphal defect on solid medium in response to many inducing conditions; however, all of these mutants filament normally in response to serum (63, 132, 154). Interestingly, although a cek1/cek1 MAP kinase mutant strain forms morphologically normal filaments in response to serum, it has a minor growth defect on serum-containing medium (63). The cek1/cek1 mutant strain also has a virulence defect that may be attributible to this growth defect (63, 100). This indicates that the Cek1 MAP kinase may function in more than one pathway or that deletion of the gene causes aberrant cross talk between distinct MAP kinase cascades, similar to the altered signaling that occurs in a fus3 mutant of S. cerevisiae. The other elements of the pathway have small but varied effects on virulence. cst20/cst20 mutant strains have a modest virulence defect in a mouse model of systemic candidiasis (154). However, hst7/hst7 and cph1/cph1 mutant strains are able to cause lethal infection in mice at rates comparable to wild-type strains (154, 164).

View larger version (15K): [in this window] [in a new window]   FIG. 8.   Signaling cascades regulating virulence of C. albicans. Two parallel signaling cascades involving a MAP kinase and a cAMP-PKA signaling pathway regulate filamentation and virulence of this human fungal pathogen. AC, adenylycyclase.

In addition to these components, a MAP kinase phosphatase, Cpp1, has been identified which regulates filamentous growth in C. albicans (62). Disruption of both alleles of the CPP1 gene derepresses hyphal production and results in a hyperfilamentous phenotype. This hyperfilamentation is suppressed by deletion of the MAP kinase Cek1 (62). cpp1/cpp1 mutant strains are also reduced for virulence in both systemic and localized models of candidiasis (62, 99).

In addition to the transcription factor Cph1 and the MAP kinase module that regulates it, a second transcription factor, Efg1, that regulates filamentous growth in C. albicans has been identified (Fig. 8) (265). Efg1 is a conserved basic helix-loop-helix-type protein that is homologous to the Phd1 transcription factor of S. cerevisiae. Similar to Phd1, heterologous overexpression of Efg1 can induce pseudohyphal growth in S. cerevisiae (265). Overexpression of Efg1 can also induce filamentous growth in C. albicans (265). An efg1/efg1 mutant strain has a moderate but not complete defect in hyphal growth in response to many environmental conditions (164). efg1/efg1 mutant strains also show an aberrant morphology in the presence of serum, forming exclusively pseudohyphae instead of the true hyphae that are produced by wild-type strains (164).

An efg1/efg1 cph1/cph1 double mutant strain has a much more severe defect in filamentous growth and does not filament under almost any conditions tested, including the presence of serum (164). In addition, while the efg1/efg1 mutant strain has a minor reduction in virulence and the cph1/cph1 mutant has little or no defect, an efg1/efg1 cph1/cph1 double mutant strain is essentially avirulent (164). Thus, Cph1 and Efg1 define elements of two separate pathways that together are essential for both filamentation and virulence in C. albicans.

A PKA-dependent pathway has been shown to regulate filamentous growth of C. albicans under some conditions (Fig. 8). An increase in cAMP levels accompanies the yeast to hyphal transition, and inhibition of the cAMP phosphodiesterase induces this transition (244). Moreover, two cell-permeating PKA inhibitors, myristoylated protein kinase inhibitor (myrPKI) (14-24)amide and the small-molecule PKA inhibitor H-89, both block hyphal growth induced by N-acetylglucosamine, but not in response to serum (42).

More recently, a PKA gene, TPK2, was identified and shown to be required for hyphal differentiation in response to a number of conditions, including the presence of serum (260). Hyphal formation in the tpk2/tpk2 mutant strain was not completely eliminated, however, and the defect was less apparent during growth at 37°C (260). Overexpression of the Tpk2 catalytic subunit also induced hyphal growth under normally noninducing conditions both in liquid culture and on solid medium (260). This is consistent with a model in which PKA acts positively in one of two or more pathways regulating dimorphism.

The phenotypes of a tpk2/tpk2 mutant strain were suppressed by overexpression of either the MAP kinase Cek1 or the transcription factor Efg1 (260). On the other hand, overexpression of Tpk2 could suppress the hyphal defect of a cek1/cek1 mutant strain but not that of an efg1/efg1 mutant strain (260). Efg1 has a single PKA consensus phosphorylation site, which may be required for its transcriptional activation activity (260). These findings suggest that the Efg1 transcription factor may lie downstream of cAMP and the PKA homolog Tpk2 in one of two major pathways regulating hyphal morphogenesis in C. albicans. Interestingly, while Efg1 plays a prominent role in regulating filamentation in C. albicans, mutation of the related protein Phd1 confers only a minor defect in S. cerevisiae filamentation (92, 164, 296).

In S. cerevisiae, the Ras2 protein regulates pseudohyphal growth and functions upstream of both the MAP kinase and cAMP-PKA pathways and, in conjunction with Ras1, is essential for viability. A single Ras homolog, Ras1, has been identified in C. albicans which is not essential for survival (81). ras1/ras1 mutant strains have a severe defect in hyphal growth in response to serum and other conditions (81). In addition, while a dominant negative Ras1 mutation (Ras1^A16) caused a defect in filamentation, a dominant active Ras1 mutation (Ras1^V13) enhanced the formation of hyphae (81). ras1/ras1 mutant strains exhibit a filamentation defect similar to that of efg1/efg1 cph1/cph1 mutant strains, and evidence from S. cerevisiae indicates that Ras2 lies upstream of both the cAMP-PKA and MAP kinase pathways regulating pseudohyphal growth (171, 201, 223, 236). These findings are consistent with a model in which Ras1 lies upstream of the related pathways in C. albicans and regulates hyphal morphogenesis. However, these studies indicate that additional pathways may also control filamentation in C. albicans, because even the ras1/ras1 and efg1/efg1 cph1/cph1 mutant strains are able to form hyphae under some conditions (81, 234).

Another signaling protein that regulates morphogenesis of C. albicans is Tup1, a homolog of the general transcriptional repressor in S. cerevisiae (33) (Fig. 8). S. cerevisiae tup1/tup1 mutant strains have multiple phenotypes, including increased flocculence, temperature-sensitive growth, and inability to sporulate. Heterologous expression of the C. albicans Tup1 protein can suppress these mutant phenotypes. S. cerevisiae tup1/tup1 mutant strains are also defective in pseudohypal growth, but this is complicated by the fact that Tup1 is required for maintenance of the diploid state in S. cerevisiae (33). In contrast, the tup1/tup1 deletion in C. albicans results in a constitutive hyphal growth phenotype under all conditions tested (33). Therefore, although Tup1 does seem to act as a general transcriptional repressor in C. albicans as it does in S. cerevisiae, it seems to have a unique function in repressing the induction of the hyphal phase in this fungus. In addition, although the Tup1 protein has been shown to be epistatic to the transcription factor Cph1, recent genetic evidence indicates that it does not lie downstream of either Cph1 or Efg1 (34, 252), but rather in an independent pathway that regulates filamentation. Furthermore, a cph1/cph1 efg1/efg1 tup1/tup1 triple mutant retains the ability to filament in response to environmental signals, providing further evidence that additional pathways regulating filamentous growth exist (34). In addition, the hyperfilamentous tup1/tup1 mutant strain has a virulence defect, which is consistent with the finding that a cpp1/cpp1 mutant strain also exhibits enhanced hyphal formation and impaired virulence. These findings support a model in which both the yeast form and the filamentous form are required to maintain a stable infection.

Although there seems to be a clear link between dimorphism and virulence in C. albicans, it is difficult to unambiguously establish this because it is possible that the signaling elements also regulate other cellular processes. One of the ways to dissect the relative contributions of these various components to the virulence of the organism is to identify the downstream effectors of these signaling molecules. One of the genes recently shown to be regulated by Efg1 is HWP1, which encodes a protein of unknown function that is homologous to GPI-anchored proteins (252). An hwp1/hwp1 mutant strain is reduced for hyphal growth under a variety of different environmental conditions (252). Expression of Hwp1 is reduced in an efg1/efg1 mutant and increased in a tup1/tup1 mutant (252). Thus, Efg1 and Tup1 regulate at least some of the same target genes and may be acting coordinately to modulate dimorphism in C. albicans. Ece1, which is expressed during cell elongation, is also not expressed in an efg1/efg1 mutant (252). However, Ece1 is not regulated by Tup1, and an ece1/ece1 mutation does not affect filamentous growth despite being regulated by Efg1 (252).

Efg1 has also been shown to regulate other morphogenic events, including chlamydospore production and phenotypic switching (259, 261). Because Efg1 is a complex signaling molecule that regulates a variety of morphogenic processes, it cannot yet be determined to what degree each of these functions of Efg1 contributes to virulence. Recent studies of single and multiple mutant strains lacking Tup1, Cph1, and Efg1 reveal that each makes a unique contribution to the regulation of genes involved in filamentous growth, suggesting that multiple pathways rather than a single central regulatory pathway are responsible (34). Further analysis of the downstream effectors of each of these transcription factors will be required to determine the relative contribution of the processes controlled by these proteins to the overall virulence of the organism.

It has long been known that C. albicans filaments in response to a variety of environmental conditions, including serum. However, recent evidence has disputed the notion that the active portion of serum that causes filamentation is albumin. Feng and colleagues have shown that the active molecule in serum is actually a serum filtrate <1 kDa in size (81). Because this molecule could not be purified, they speculated that it may be multiple small molecules that act coordinately to induce this response. Several small molecules that regulate filamentous growth of S. cerevisiae have recently been identified, including glucose and other carbohydrates, nitrogen sources, short-chain alcohols, and mating pheromone (168, 169, 172, 235).

Although C. albicans is diploid and was thought to be an asexual organism, several lines of evidence suggested that a cryptic sexual cycle could exist. First, it was shown some time ago that DNA exchange could be forced between strains of C. albicans following mild UV irradiation and that these events involved recombination rather than merely the formation of heterokaryons (268). Furthermore, more recently, Graser and colleagues used population genetics to show that the population structure of C. albicans suggests that some form of recombination is occurring within this species (97). Several homologs of genes regulating mating in S. cerevisiae have been identified in C. albicans. In addition to a G protein  subunit homolog and the MAP kinase cascade components, a homolog of the Ste6 protein which transports a-factor pheromone in S. cerevisiae has been identified in C. albicans (230).

Recently, regions of the C. albicans genome resembling the MATa and MAT mating type loci in S. cerevisiae have been discovered and named the MTL and MTLa loci (MTL stands for mating type-like) (116). The two chromosomes are only 48% identical in this region but greater than 99% identical in the rest of the genome, findings that are consistent with other MAT loci that have been identified (116). In addition, this locus contains homologs of the a1, 1, and 2 genes that regulate mating type in S. cerevisiae, and these genes are arranged in the same manner as in S. cerevisiae (116). In S. cerevisiae, the diploid state is maintained by the action of the a1/2 protein complex, which represses the expression of haploid-specific genes. Evidence suggests that the a1/2 homolog functions similarly in C. albicans. The heterotrimeric G protein  subunit homolog gene CAG1 has a consensus binding site for the a1/2 complex. Moreover, expression of the CAG1 gene in either S. cerevisiae or C. albicans has been shown to be regulated by the a1 and 2 proteins (116).

Recent studies by two independent groups have demonstrated that mating can occur in C. albicans. Magee and Magee engineered MTLa/MTLa and MTL/MTL homozygous strains by inducing loss and reduplication of chromosome V by selection for growth on sorbose (183). With appropriately marked auxotrophic strains, they observed the production of prototrophic tetraploid strains following coincubation. Independently, Hull et al. engineered strains with mutations in the a1 or 1 and 2 genes of the mating type-like loci (117). In this case, mating was observed following coinfection of mice with marked strains and again gave rise to prototrophic tetraploid progeny. In these later studies, no mating has thus far been detected in vitro, suggesting that strain differences may contribute to the ability to detect mating in vitro. These studies overturn the long-held dogma that C. albicans is asexual and open the door to further genetic analysis in this important human fungal pathogen. An important next step will be to determine whether these tetraploid products of mating can be induced to undergo meiosis and sporulate and to establish where in vivo mating occurs and whether there is an impact on virulence.

C. neoformans is a basidiomycetous fungus and a significant human pathogen with worldwide distribution. Its importance as an opportunistic pathogen has increased in the past two decades, largely as a result of AIDS, cancer chemotherapy, and immunosuppression for organ transplants (193). Infections begin in the lung following inhalation of small, dessicated yeast cells or spores, both of which are small enough t