Snf1 Kinases with Different {beta}-Subunit Isoforms Play Distinct Roles in Regulating Haploid Invasive Growth

The Snf1 protein kinase of Saccharomyces cerevisiae has beenshown to have a role in regulating haploid invasive growth inresponse to glucose depletion. Cells contain three forms ofthe Snf1 kinase, each with a different ß-subunit isoform,either Gal83, Sip1, or Sip2. We present evidence that differentSnf1 kinases play distinct roles in two aspects of invasivegrowth, namely, adherence to the agar substrate and filamentation.The Snf1-Gal83 form of the kinase is required for adherence,whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation.Genetic evidence indicates that Snf1-Gal83 affects adherenceby antagonizing Nrg1- and Nrg2-mediated repression of the FLO11flocculin and adhesin gene. In contrast, the mechanism(s) bywhich Snf1-Gal83 and Snf1-Sip2 affect filamentation is independentof FLO11. Thus, the Snf1 kinase regulates invasive growth byat least two distinct mechanisms.

INTRODUCTION

Top

Introduction

In fungi such as Candida albicans, Cryptococcus neoformans,and Ustilago maydis, the ability of cells to undergo a dimorphictransition between yeast-like growth and filamentous or hyphalgrowth is an important determinant of pathogenicity. The buddingyeast Saccharomyces cerevisiae also exhibits a dimorphic transitionin response to nutrient limitation and provides a convenientgenetic system for studying this process. The nature and regulationof the dimorphic transition is determined by ploidy (for reviews,see references 10, 22, and 28). Haploid S. cerevisiae cellsinitiate filamentous invasive growth upon glucose depletion(7, 37), whereas diploid cells make a transition to pseudohyphalgrowth in response to nitrogen starvation (12), although filamentousgrowth can also occur in response to carbon source depletion(8, 21). The distinct form of pseudohyphal growth that occursin mutants lacking the forkhead transcription factors, whichcontrol cell cycle-regulated genes, is nutrient independent(15, 51).

Haploid invasive growth depends on FLO11, a gene encoding acell surface glycoprotein that functions as a flocculin or adhesin(13, 21, 24, 25, 36). FLO11 has a large and complex promoterthat is regulated by the cyclic AMP-dependent protein kinaseand mitogen-activated protein kinase pathways (24, 27, 32, 38,40). The Snf1 kinase, which is required for haploid invasivegrowth (7), also regulates FLO11 expression in response to glucosedepletion by antagonizing Nrg1- and Nrg2-mediated repressionof FLO11 (20, 47). One aspect of invasive growth is adherenceto the support (36), and Snf1 and the Nrg repressors correspondinglyaffect the Flo11-dependent adherence of cells to a plastic surface(20). Another aspect of invasive growth is filamentation, whichentails cell elongation and a switch from axial to unipolarbudding; these morphological changes also require Snf1 (7).

The Snf1 kinase is highly conserved in fungi, plants, and animals(called AMP-activated kinase in mammals), and this family ofkinases has broad roles in transcriptional and metabolic regulationin response to stress (for reviews, see references 14 and 19).In the pathogenic yeast C. albicans, Snf1 function is essentialfor viability (9, 34). In S. cerevisiae, Snf1 is required formany aspects of transcriptional and metabolic adaptation toglucose limitation (5, 11) and has been implicated in otherstress responses (1, 43). Besides haploid invasive growth, Snf1also affects developmental processes such as diploid pseudohyphalgrowth (20), aging (3, 23), and meiosis and sporulation.

The participation of Snf1 in diverse regulatory responses inS. cerevisiae is facilitated by the existence of multiple formsof the kinase, as is also the case in mammals. S. cerevisiaecells contain three forms, each comprising the catalytic subunitSnf1, the activating subunit Snf4, and one of three ß-subunitisoforms, Gal83, Sip1, or Sip2 (17, 49). We will refer to theseforms by designating the ß subunit, for example, Snf1-Gal83.Although the ß subunits exhibit significant functionalredundancy, they also have important roles in regulating thespecificity of the kinase (3, 42, 45, 49); for example, Gal83mediates the physical interaction of the kinase with Sip4, aSnf1-dependent transcriptional activator of gluconeogenic genes(45). The ß subunits also regulate the subcellularlocalization of the kinase and presumably its access to differentsubstrates. All of the ß subunits are cytoplasmicwhen cells are grown in abundant glucose; upon glucose depletion,Gal83 directs Snf1 to the nucleus, Sip1 is relocalized apparentlyto membranes and then to the vacuole, and Sip2 remains cytoplasmic(46). Finally, at least one kinase form is subject to multipleregulatory inputs: Snf1-Gal83 is regulated both by the glucosesignaling pathway that inhibits its catalytic activity and bya distinct pathway that controls its localization in responseto fermentable carbon sources (46). Thus, the ß subunitsboth confer specificity and provide versatility in the controlof different functions of Snf1.

To explore the functions of the Snf1 kinase in regulating invasivegrowth, we have examined the roles of different Snf1 kinasesin adherence to the support and filamentation. We present evidencethat Snf1 affects adherence by a pathway involving the Snf1-Gal83form, the Nrg repressors, and FLO11. In contrast, both Snf1-Gal83and Snf1-Sip2 affect filamentation by a FLO11-independent pathway(s).Thus, these studies reveal two distinct mechanisms for regulationof invasive growth by the Snf1 kinase.

MATERIALS AND METHODS

Top

Materials and Methods

Strains and genetic methods. S. cerevisiae strains used in this work are listed in Table1. All strains were in the ${Sigma}$ 1278b genetic background and werederived by standard genetic methods (39) from the isogenic strainsMY1401, MY1402, and MY1411 of the Sigma2000 series (Microbia,Cambridge, Mass.). gal83 ${Delta}$ ::KlURA3 is a complete disruption ofthe GAL83 coding sequence created by homologous recombinationwith the direct repeat-flanked Kluyveromyces lactis URA3 marker,using the method of adaptamer-directed gene disruption (35).Popout recombinants of gal83 ${Delta}$ ::KlURA3 were selected on 5-fluorooroticacid to generate gal83 ${Delta}$ . sip1 ${Delta}$ ::KanMX6 (46) and snf1 ${Delta}$ 10 (6) wereintroduced as described previously. sip2::KanMX6 is a completedeletion of the SIP2 coding sequence created by homologous recombinationwith the KanMX6 marker by using the method of PCR synthesisof marker cassettes (48). The FLO11 upstream region was replacedwith the Schizosaccharomyces pombe adh⁺ promoter as describedpreviously (31), using promoter sequence amplified from plasmidspADH-CLB2 (2), to generate the SpADHp-FLO11 allele. All mutationswere first introduced into MY1401, and combinations of thesealleles were then obtained by genetic crossing. Genotypes wereestablished by mutant phenotypes and by analysis of genomicDNA using PCR. Complete synthetic medium (CSM) was purchasedcommercially (BIO 101 Systems; Q-BIOgene, Carlsbad, Calif.),and rich medium was yeast extract peptone (YEP).

View this table:
[in this window]
[in a new window]
TABLE 1. S. cerevisiae strains used in this study

Invasive growth assay. The plate washing assay described by Roberts and Fink (37) wasmodified as follows. Fresh colonies from YEP-plus-2% glucoseplates were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA,pH 7.5), or fresh overnight cultures were grown in YEP plus2% glucose and cells were collected, washed, and resuspendedin TE buffer; both methods gave similar results in invasivegrowth assays. Cells were spotted onto CSM containing 0.1% glucoseand 2% agar, incubated at 26°C for 1 to 4 days, and photographed.Plates were washed under a stream of water by rubbing with abent glass rod and then photographed again. Invasion was alsoassayed on YEP containing 2% glucose and 2.5% agar.

Assay for adherence to plastic. Assays for adherence to the wells of a polystyrene 96-well microtiterplate (Falcon Microtest flat bottom plate, 35-1172; Becton-DickinsonLabware) were carried out as described elsewhere (36). Cellswere grown in CSM plus 2% glucose to an optical density at 600nm (OD₆₀₀) of 0.5 to 1.5, collected, washed, and resuspendedto an OD₆₀₀ of 1 in CSM with 2% or 0.1% glucose. Cells (0.1ml) were transferred to the wells of a microtiter plate andincubated at 30°C for 1 to 7.5 h, as indicated. The cellswere then stained with crystal violet, and the wells were washedrepeatedly with water.

Microscopy. Cells were grown to stationary phase in YEP plus 2% glucoseliquid medium, washed twice with TE buffer, diluted, and spreadat low concentration onto CSM with 0.1% or no glucose; 0.1%glucose was as repressive for filamentation as 2% glucose, butoverall growth was more closely matched to that occurring onmedium with no added glucose. Plates were incubated at 26°Cfor 16 to 24 h, and microcolonies were viewed using a NikonEclipse E800 fluorescence microscope. Images were taken withan Orca100 (Hamamatsu) camera, using Open Lab (Improvision)software, and processed in Adobe Photoshop 5.5.

RESULTS

Top

Results

Gal83 is required for invasive growth. To test the requirement for the different ß subunitsin invasive growth, we examined isogenic sip1 ${Delta}$ , sip2 ${Delta}$ , and gal83 ${Delta}$ mutants in the ${Sigma}$ 1278b genetic background. Cell suspensions werespotted on solid CSM containing a low concentration of glucose(0.1%); previously, Cullen and Sprague showed that invasionoccurs in response to glucose limitation (7). After incubationfor 2 days, the plates were washed. Cells that remained on theplate, having adhered to or penetrated the agar, are describedhere as exhibiting invasive growth (Fig. 1A). The gal83 mutantwas markedly defective, as were the snf1 and gal83 sip1 sip2mutants. The sip1 mutant resembled the wild type, as reportedpreviously (7), as did the sip2 mutant. Although the snf1 andgal83 sip1 sip2 strains grew poorly on 0.1% glucose, the gal83mutant grew as well as the wild type; hence, there was no correlationbetween the extent of growth and invasion.

View larger version (51K):
[in this window]
[in a new window]
FIG. 1. Assay for invasive growth. Cells were grown on solid CSM plus 0.1% glucose (A and B) or on YEP plus 2% glucose containing 2.5% agar (B) for 2 days at 26°C and photographed. Plates were washed and photographed again. The wild-type strain was MCY4565 and mutant strains (listed in Table 1) were also all MATa and carried the same auxotrophic markers (in particular, all are Ura⁺), so that invasiveness would not be affected by differences in genetic background. On YEP plus 2% glucose, invasive growth of the wild type is easily detectable by 3 days.

Because the ß subunits exhibit significant functionalredundancy, we also constructed double mutants (Fig. 1A). Thesip1 sip2 double mutant exhibited invasive growth, indicatingthat the Gal83 subunit is both necessary and sufficient. Thegal83 sip1 mutant was noticeably more proficient in invasivegrowth than the gal83 single mutant, and the gal83 sip2 sip1strain was more proficient than the gal83 sip2 strain; thesefindings suggest that Sip1 inhibits this process, either bya direct mechanism or perhaps indirectly through effects onthe metabolic state of the cell. The sip2 mutation slightlyreduced invasiveness of both gal83 and gal83 sip1 strains, suggestingthat Sip2 has an auxiliary positive role in invasive growth.A positive effect of Sip2 was similarly apparent in a reg1 gal83mutant background (see below) (Fig. 1B). This evidence thatthe Sip2 subunit contributes in some manner to invasive growthwas confirmed by subsequent experiments revealing a role infilamentation (see below). Together, these findings indicatethat the Snf1-Gal83 kinase has the primary role in invasivegrowth, but Sip1 and Sip2 also affect this process.

We also assayed invasive growth on YEP plus 2% glucose, whichis commonly used for such assays; however, the snf1 and gal83sip1 sip2 mutants showed a less reproducible defect than onCSM with 0.1% glucose or YEP with 0.1% glucose (data not shown).Invasive growth of the wild type occurs more rapidly on 0.1%glucose than on 2% glucose (Fig. 1B). We suggest that on 0.1%glucose, the Snf1 kinase is critical for an immediate invasiveresponse, whereas during growth on 2% glucose, wild-type andmutant cells differ in their capacity for metabolic adaptationto glucose depletion, which in turn affects the time courseof invasion. In previous studies, a snf1::URA3 mutant exhibiteda pronounced defect in invasiveness on YEP plus 2% glucose relativeto the SNF1 ura3 parent (7), but the magnitude of the defectmay have been enhanced by the auxotrophic difference because,in our strains, the ura3 mutation improves invasive growth (datanot shown).

To confirm that Snf1-Gal83 is required for invasive growth onYEP plus 2% glucose, we took advantage of our previous findingthat a reg1 mutant is much more invasive than the wild typeon this medium (20). Reg1 directs protein phosphatase type 1to inhibit the Snf1 kinase (16, 26, 29, 41, 44), and mutationof REG1 relieves glucose repression of many genes, includingFLO11 (20). The effect of reg1 on invasive growth requires theSnf1 kinase (20) and presumably reflects the relief of glucoseinhibition of the kinase, because the reg1 mutant was more invasivethan wild type on 2% glucose but not on 0.1% glucose (Fig. 1B).The gal83 mutation caused a defect in invasive growth of thereg1 mutant on both YEP plus 2% glucose and CSM with 0.1% glucose;moreover, sip2 further reduced invasion of the reg1 gal83 mutanton both media. These findings exclude the possibility that theeffects of gal83 and sip2 are specific to a particular growthmedium.

Gal83 affects adherence to plastic. The ability of yeast cells to adhere to a plastic surface isrelated to their capacity for invasive growth in that both processesrequire the Flo11 flocculin and adhesin, which is essentialfor adherence to the support (13, 36), and both are dependenton Snf1 kinase activity (20). We therefore tested the role ofGal83 in adherence to plastic. Wild-type and gal83 mutant strainswere grown to exponential phase in 2% glucose, resuspended in0.1% glucose, and transferred to the wells of a microtiter plate.The mutant cells adhered to the plastic less well than the wild-typecells, although the difference was not striking (Fig. 2A). Wealso tested strains carrying the reg1 mutation, which enhancesplastic adherence (dependent on the Snf1 kinase and FLO11 [20])and therefore improves the sensitivity of the assay. The reg1gal83 mutant was clearly defective in adherence relative tothe reg1 strain and resembled the reg1 snf1 and reg1 sip1 sip2gal83 strains (Fig. 2B). Thus, Snf1-Gal83 has a role in adherence.

View larger version (41K):
[in this window]
[in a new window]
FIG. 2. Assay for adherence to plastic. Cells were assayed for adherence to polystyrene (36) as described in Materials and Methods. Cells were resuspended in CSM with 2 or 0.1% glucose, as indicated, and transferred to the wells of a microtiter plate. (A) Strains were MCY4702, MCY4704, and MCY4712; all were MATa. Cells were incubated for the indicated time before washing. (B) The wild-type strain was MCY4565, and mutant strains were also all MATa and carried the same auxotrophic markers. Cells were incubated for 7.5 h before washing. Assays were carried out in quadruplicate.

Evidence that Snf1-Gal83 antagonizes Nrg1 and Nrg2, two repressors of FLO11. Previous studies showed that one of the roles of the Snf1 kinasein regulating invasive growth is antagonism of the zinc-fingerproteins Nrg1 and Nrg2, which repress FLO11 (20). Both Nrg repressorsinteract physically with Snf1 (47), and mutation of NRG1 andNRG2 alleviates snf1 mutant defects in invasive growth, adherenceto plastic, and expression of the STA2 (glucoamylase) promoter(20), which is nearly identical to that of FLO11 for 3.5 kb.To determine whether the Snf1-Gal83 form of the kinase is responsiblefor antagonism of Nrg-mediated repression, we examined the geneticrelationships of nrg1 and nrg2 to gal83 with respect to thesephenotypes. First, we assayed invasive growth; in a gal83 mutant,each single nrg mutation had some effect and both nrg mutationstogether strongly restored invasive growth (Fig. 3). The nrgmutations also alleviated the gal83 mutant defect in adherenceto plastic (Fig. 2A). Finally, we assayed expression of lacZfused to the STA2 promoter in ß subunit mutants duringgrowth in glucose and after a shift to low (0.05%) glucose.Derepression of STA2-lacZ was greatly reduced by gal83 but wasnot affected by mutation of the other ß subunits (Fig.4A), and expression was restored in a gal83 nrg1 nrg2 triplemutant (Fig. 4B). These data implicate Snf1-Gal83 in reliefof glucose repression by the Nrg proteins. Taken together, thesegenetic findings support the view that the Gal83 subunit mediatesthe functional interaction of the Snf1 kinase with Nrg1 andNrg2.

View larger version (39K):
[in this window]
[in a new window]
FIG. 3. Mutation of NRG1 and NRG2 restores invasive growth in a gal83 mutant. Wild-type (GAL83) and gal83 ${Delta}$ strains carried nrg1 and nrg2 mutations, singly and in combination, as indicated. Cells were spotted on YEP plus 2% glucose containing 2.5% agar, incubated at 30°C for 2 days, and washed. Strains tested were MATa (MCY4700 series in Table 1). The gal83 mutants all carry the K. lactis URA3 marker. +, wild-type NRG alleles.

View larger version (24K):
[in this window]
[in a new window]
FIG. 4. The gal83 and nrg mutations affect STA2-lacZ expression. Strains with the indicated genotype were transformed with pLCLG-Staf, a centromeric plasmid carrying STA2-lacZ (18). The STA2 promoter is nearly identical to the FLO11 promoter for 3.5 kb, except for two 20- and 64-bp insertions (21). (A) Strains were those shown in Fig. 1. Transformants were grown to mid-log phase in CSM plus 2% glucose lacking leucine to select for the plasmid and were shifted to CSM plus 0.05% glucose for the indicated times. ß-Galactosidase activity was assayed in permeabilized cells and expressed in Miller units, as described previously (47). Values are average activity for four transformants, and standard errors are indicated. (B) Strains were those shown in Fig. 3. Transformants were grown overnight in selective CSM plus 2% glucose, diluted to an OD₆₀₀ of 0.1 in YEP plus 2% glucose, and grown to an OD₆₀₀ of 0.5. An aliquot of cells was shifted to YEP plus 0.05% glucose for 180 min. Assays were as described for panel A.

Sip2 and Gal83 are each sufficient for filamentation. We next tested the role of different Snf1 kinases in filamentation,which entails cell elongation and a switch from axial to unipolarbudding. Filamentation occurs in response to low glucose, anda snf1 mutant is defective in both elongation and unipolar budding(7). We used the assay devised by Cullen and Sprague (7) toobserve morphological changes. Cells were spread onto CSM lackingglucose and allowed to form microcolonies. Microscopic examinationof the ß subunit mutants showed that each single mutantwas fully competent for filamentation, as were the sip1 sip2and sip1 gal83 double mutants (Fig. 5A). The sip2 gal83 doublemutant, however, was defective and resembled the snf1 and sip1sip2 gal83 mutants (Fig. 5A).

View larger version (82K):
[in this window]
[in a new window]
FIG. 5. Microscopic examination of mutants. Cells were spread onto CSM lacking glucose (A) or CSM plus 0.1% glucose (B) and incubated for 16 to 24 h at 26°C to form microcolonies as described in Materials and Methods. Strains were those shown in Fig. 1 and other auxotrophically matched reg1 strains. Bars, 10 µm.

We further examined the effects of a reg1 mutation. The reg1mutant exhibited filamentous growth on medium containing no,0.1, or 2% glucose (Fig. 5B and data not shown), consistentwith its other glucose-insensitive phenotypes. However, reg1snf1 cells did not filament, indicating that the enhanced filamentationdepends on Snf1 activity. This finding allowed us to test thephenotypes caused by ß subunit mutations on mediumcontaining glucose and thereby rule out the possibility thatthe impaired filamentation of sip2 gal83 cells results fromtheir poor growth in the absence of glucose. The combinationsgal83 sip2 and gal83 sip1 sip2 impaired filamentation in thereg1 mutant background, whereas other combinations had no noticeableeffect (Fig. 5B). These findings indicate that either Gal83or Sip2 is sufficient for filamentation.

The role of Sip2 in filamentation most likely accounts for theobservation that mutation of SIP2 in a gal83 strain furtherdiminishes invasive growth (Fig. 1). The following evidencealso suggests that Snf1-dependent filamentation contributesto invasive growth. Replacement of the endogenous FLO11 promoterwith the S. pombe adh⁺ promoter was shown to give constitutive,low-level FLO11 expression and to abolish invasion of wild-typecells (31); in our strain background, this replacement similarlyabolished invasive growth (Fig. 6B). However, reg1 mutant cellscarrying the SpADHp-FLO11 allele filamented in the presenceof glucose, unlike wild-type cells (Fig. 6A), and exhibiteddetectable invasive growth on agar (Fig. 6B). Although it ispossible that reg1 upregulates other genes besides FLO11 toaffect adherence to agar, these findings are consistent withthe idea that Snf1-dependent filamentation contributes to invasiveness.

View larger version (52K):
[in this window]
[in a new window]
FIG. 6. Agar invasion and filamentation of strains expressing FLO11 from the S. pombe adh⁺ promoter. (A) Wild-type and reg1 mutant strains carrying the SpADHp-FLO11 allele were spread onto CSM containing 0.1% or no glucose and allowed to form microcolonies as described in Materials and Methods. (B) Strains of the indicated genotypes were tested for invasive growth on YEP plus 2% glucose with 2.5% agar at 26°C. Plates were photographed after 10 days. The REG1 SpADHp-FLO11 strain should have a slight advantage for invasive growth because it carries the ura3 mutation; nonetheless, the reg1 ${Delta}$ ::URA3 SpADHp-FLO11 strain was more invasive. (C) Strains of the indicated genotypes were examined as described for panel A. Bars, 10 µm.

If Snf1-Gal83 and Snf1-Sip2 affect filamentation through different,redundant pathways, then Snf1-Gal83 could conceivably promotefilamentation through the same pathway by which it affects adherence.To address this possibility, we examined the roles of the Nrgrepressors and FLO11 in filamentation. The nrg1 nrg2 doublemutation did not significantly affect filamentation in an otherwisewild-type genetic background or suppress the filamentation defectof a snf1 mutant on medium containing 0.1% or no glucose (datanot shown). Previous studies showed that diploid filamentationrequires FLO11 (24, 40); in contrast, we found that in haploids,FLO11 is dispensable, as both flo11 ${Delta}$ and reg1 flo11 ${Delta}$ mutant cellsexhibited filamentous morphology similar to that of the correspondingFLO11 strains in either 0.1% or no glucose (Fig. 6C). Theseresults are consistent with recent evidence that flo11 doesnot affect haploid filamentous morphology in another strainbackground (8). Thus, the filamentation pathway involving Snf1-Gal83is distinct from the pathway by which Snf1-Gal83 affects adherenceto agar and plastic.

DISCUSSION

Top

Discussion

We have investigated the roles of the Snf1 kinase in regulatinghaploid invasive growth in response to glucose depletion. Weshow that different forms of the kinase are required for twodifferent aspects of invasive growth, namely, adherence to theagar substrate and filamentation. The Snf1-Gal83 form of thekinase affects adherence by a mechanism involving the Nrg repressorsand FLO11. The Snf1-Gal83 and Snf1-Sip2 kinases together affectcell morphology by a mechanism(s) that is independent of Nrgproteins and FLO11. Thus, the Snf1 kinase has multiple rolesin the complex process of invasive growth (Fig. 7). These findingsalso provide further evidence that the different ßsubunits confer specificity to the Snf1 kinase in cellular regulatoryresponses.

View larger version (18K):
[in this window]
[in a new window]
FIG. 7. Model for roles of the Snf1 kinase in invasive growth. The Snf1-Gal83 form of the kinase regulates FLO11 and possibly other Nrg-repressed genes that affect adhesion. Snf1 may also affect FLO11 expression by other mechanisms. Snf1-Gal83 and Snf1-Sip2 affect filamentation by a pathway(s) that does not involve FLO11 or Nrg repressors. (A) These two kinase forms may function redundantly in the same pathway. (B) Alternatively, the two kinases may function in two distinct pathways, either of which suffices for filamentation. Moreover, the role of Snf1 in filamentation may involve multiple targets. Snf1-Sip1 also has an inhibitory effect on invasive growth, but the mechanism is not yet understood.

We first showed that the effects of Snf1 on invasive growthare mediated primarily by Snf1-Gal83. Gal83 is both necessaryand sufficient for invasive growth, as a gal83 mutant was defectiveand a sip1 sip2 strain was proficient. However, the sip2 mutationcaused a noticeable decrease in invasive growth in a gal83 mutantbackground, which we attribute to the defect in filamentation.In contrast, mutation of SIP1 improved invasive growth in agal83 mutant, suggesting an inhibitory role for Sip1. Snf1-Sip1may directly inhibit some function that promotes invasive growthor may act indirectly through effects on the metabolic stateof the cell. We think it unlikely that loss of Sip1 simply affectsthe distribution of the Snf1 catalytic subunit among the differentisoforms because Sip1 is not an abundant ß subunitand because loss of Sip1 has an effect in a gal83 sip2 mutant.

Genetic evidence indicates that the effects of Snf1-Gal83 onadherence occur, at least in part, by inhibition of the Nrg1and Nrg2 repressors. These repressors may in fact be the majortargets, because adherence to both agar and plastic substrateswas effectively restored in a gal83 mutant by the double nrg1nrg2 mutation. Snf1 interacts physically with Nrg1 and Nrg2(47), and one possibility is that Gal83 interacts directly withthese repressors, as it does with the activator Sip4 (45). Wewere unable to detect two-hybrid interaction of Gal83 with Nrg1or Nrg2 in several different assays (V. K. Vyas and C. D. Berkey,unpublished results), but these negative results do not excludethe possibility that Gal83 contributes to the Snf1-Nrg interaction.Alternatively, Gal83 could simply facilitate the interactionbetween Snf1 and these repressors by virtue of its role in thenuclear localization and nuclear function of the kinase (46).Gal83 is the ß subunit that is primarily responsiblefor the nuclear localization of Snf1 upon glucose depletion(46), and Nrg1 is a nuclear protein (47).

We present evidence that one of the functional targets of theSnf1-Gal83/Nrg pathway is FLO11, which is essential for invasivegrowth and adherence to plastic (21, 24, 25, 36). This pathwaymay also regulate other genes needed for invasive growth, asonly a few Nrg1- and Nrg2-repressed genes besides FLO11 havebeen identified (20, 33, 47, 50). It is noteworthy that an orthologof the Nrg proteins in the fungal pathogen C. albicans has beenshown to repress filamentous growth and expression of the hypha-specificadhesin genes HWP1, ALS3, and ALS8 (4, 30).

We also examined the role of the Snf1 kinase in promoting filamentationand found that either the Snf1-Gal83 or Snf1-Sip2 form is sufficient.The simple model is that the Snf1 kinase affects filamentationby a pathway in which both Snf1-Gal83 and Snf1-Sip2 can participate,such that the presence of either one alone suffices for functionof the pathway (Fig. 7A). Although Gal83 becomes enriched inthe nucleus in response to glucose depletion while Sip2 remainspredominantly cytosolic (46), their different localizationsdo not preclude performance of an identical function. Alternatively,the Snf1 kinase could promote filamentation by two redundantpathways, one involving Snf1-Gal83 and the other Snf1-Sip2;however, Snf1-Gal83 must act through a pathway different fromthat by which it regulates adherence (Fig. 7B). The function(s)of the Snf1 kinase in filamentation could potentially involvetranscriptional control or regulation of metabolic enzymes orother cellular components. Very recent work indicates that onefunction of Snf1 is to promote the disappearance of the axialbudding determinant Axl1 in response to glucose limitation (8).

This study thus identifies at least two distinct signaling mechanismsby which the Snf1 kinase contributes to control of two aspectsof invasive growth. It is likely that Snf1 participates in yetother regulatory interactions besides those represented in Fig.7; for example, Snf1-Gal83 may relieve Nrg-mediated repressionof other genes besides FLO11, as mentioned above. The involvementof both Snf1-Gal83 and Snf1-Sip2 also raises the possibilitythat adherence and filamentation are differentially regulatedby nutrient signals. A glucose signaling pathway inhibits theSnf1 catalytic activity, but a distinct pathway regulates thenucleocytoplasmic distribution of Snf1-Gal83 in response tofermentable carbon sources, whereas Snf1-Sip2 is constitutivelycytoplasmic (46). Further studies are needed to resolve thevarious roles of Snf1 in the very complex regulatory networkthat controls invasive growth.

ACKNOWLEDGMENTS

We thank A. Amon, A. Wach, R. Rothstein, and R. Reid for plasmids.

This work was supported by NIH grant GM34095 to M.C.

FOOTNOTES

* Corresponding author. Mailing address: 701 W. 168th St., HSC922, New York, NY 10032. Phone: (212) 305-6314. Fax: (212) 305-1741. E-mail: mbc1{at}columbia.edu.

REFERENCES

Top