Role of Tos3, a Snf1 Protein Kinase Kinase, during Growth of Saccharomyces cerevisiae on Nonfermentable Carbon Sources

In Saccharomyces cerevisiae, Snf1 protein kinase of the Snf1/AMP-activatedprotein kinase family is required for growth on nonfermentablecarbon sources and nonpreferred sugars. Three kinases, Pak1,Elm1, and Tos3, activate Snf1 by phosphorylation of its activation-loopthreonine, and the absence of all three causes the Snf^–phenotype. No phenotype has previously been reported for thetos3 ${Delta}$ single mutation. We show here that, when cells are grownon glycerol-ethanol, tos3 ${Delta}$ reduces growth rate, Snf1 catalyticactivity, and activation of the Snf1-dependent carbon source-responsiveelement (CSRE) in the promoters of gluconeogenic genes. In contrast,tos3 ${Delta}$ did not significantly affect Snf1 catalytic activity orCSRE function during abrupt glucose depletion, indicating thatTos3 has a more substantial role in activating Snf1 proteinkinase during growth on a nonfermentable carbon source thanduring acute carbon stress. We also report that Tos3 is localizedin the cytosol during growth in either glucose or glycerol-ethanol.These findings lend support to the idea that the Snf1 proteinkinase kinases make different contributions to cellular regulationunder different growth conditions.

INTRODUCTION

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Introduction

Snf1 protein kinase of the yeast Saccharomyces cerevisiae isa member of the Snf1/AMP-activated protein kinase (AMPK) familyof metabolic stress response kinases (5, 12, 18). In mammals,AMPK is activated by stresses that deplete ATP and by hormones,including leptin and adiponectin (23, 42), and serves as a majorregulator of lipid and glucose metabolism. In yeast, Snf1 proteinkinase is also activated by various stresses, notably glucoselimitation (17, 22, 39, 41). Snf1 protein kinase regulates transcriptionand the activity of metabolic enzymes, and Snf1 is requiredfor growth on nonfermentable carbon sources and such nonpreferredsugars as sucrose, raffinose, and galactose (4, 9). Snf1 alsoaffects meiosis and sporulation, aging (1), haploid invasivegrowth (7), and diploid pseudohyphal growth (19).

Snf1 protein kinase, like AMPK, exists in three heterotrimericforms containing the Snf1 ( ${alpha}$ ) catalytic subunit; the Snf4 ( ${gamma}$ )regulatory subunit; and one of the ß subunits, Gal83,Sip1, and Sip2 (43), referred to henceforth as Snf1-Gal83, Snf1-Sip1,and Snf1-Sip2, respectively. The three ß subunitshave largely overlapping functions, and all three must be mutatedto confer the Snf^– phenotype (29); however, they exhibitdistinct subcellular localizations (15, 36) and play distinctroles in various cellular processes (1, 20, 29, 34, 37).

Three kinases that activate Snf1 by phosphorylation of its activation-loopthreonine have been identified: Pak1, Tos3, and Elm1 (16, 24,32). All three Snf1 protein kinase kinases contribute to activationof Snf1 in vivo, as the cognate genes for all three must bedeleted to confer the Snf^– phenotype of failure to growon nonpreferred carbon sources (16, 32). In addition, Elm1 hasroles in controlling cell morphology, cell cycle progression,and filamentous invasive growth (2, 3, 7, 30, 31) that are notknown to be related to Snf1 function. This kinase cascade isconserved in mammals, where the tumor suppressor kinase LKB1,an ortholog of Pak1, Tos3, and Elm1, phosphorylates and activatesAMPK (13, 16, 40).

Why do yeast cells have three different activating kinases forSnf1? There is some evidence to suggest that the different kinasesplay distinct roles with respect to regulation of Snf1 function.In particular, Pak1 has a specific role in the nuclear enrichmentof Snf1-Gal83 (14), which is the only form of the kinase thatbecomes enriched in the nucleus in response to glucose limitation(36). In a pak1 ${Delta}$ mutant, nuclear enrichment is defective followingsudden glucose depletion and during long-term growth in glycerol-ethanolor galactose (14). This enrichment requires phosphorylationof the activation-loop Thr of Snf1, but it is not clear whyPak1 is specifically required. Previously, we excluded the possibilitythat each kinase is dedicated to activation of a specific formof Snf1 protein kinase; both Pak1 and Elm1 activated both Snf1-Gal83and Snf1-Sip2, as judged by assays of Snf1 catalytic activityin vitro and by growth phenotypes (14). These findings ruleout exclusivity, but the activating kinases may exhibit somepreferential recognition of different Snf1 protein kinase forms.

Somewhat surprisingly, the tos3 ${Delta}$ mutation caused no phenotypein previous assays (14). When glucose-grown cells were subjectedto acute glucose deprivation, Pak1 was the most important ofthe three with respect to activating Snf1 catalytic activity,and Elm1 contributed significantly, but Tos3 had little or norole. When cells expressing only Snf1-Gal83 or Snf1-Sip2 weregrown on raffinose, the absence of Pak1 or Elm1 diminished growth,but Tos3 had no discernible role in cells expressing only Snf1-Gal83,Snf1-Sip2, or Snf1-Sip1. Perhaps Tos3 is simply a minor Snf1protein kinase kinase activity in vivo, despite its robust catalyticactivity towards recombinant Snf1 in vitro (16). However, itis also possible that one explanation for the existence of threeSnf1-activating kinases is that the different kinases play rolesunder distinct growth conditions; according to this idea, Tos3may play a more important role in regulating Snf1 under differentgrowth conditions.

In this study, we have addressed the possibility that Tos3 hasa more substantial role in activating Snf1 during growth onnonfermentable carbon sources, which is important for yeastcells in natural environments. During growth of cells on glucoseor other fermentable carbon sources, the cells first exhaustthe glucose, accumulating ethanol and other metabolic products.The cells then make a transition to respiratory growth duringthe diauxic shift and continue to double slowly until they enterstationary phase. Using several assays, we show that the tos3 ${Delta}$ mutation causes defects during growth on ethanol plus glycerol.We also assess the expression and subcellular localization ofTos3 under these conditions.

MATERIALS AND METHODS

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Materials and Methods

Strains and media. Table 1 lists S. cerevisiae strains used in this work. All strainshave the W303 genetic background. To insert the tandem affinitypurification (TAP) tag (27) at the 3' end of the TOS3 open readingframe, a DNA fragment encoding the TAP tag and the Kluyveromyceslactis URA3 marker was amplified from plasmid pBS1539 (25) withprimers containing appropriate TOS3 sequences. The purifiedPCR fragment was used to transform strain W303-1A to producestrain MCY5533. To construct MCY5510, we used a PCR fragmentamplified from a strain carrying SIP2::TAP-HI3MX6 (Open Biosystems,Huntsville, AL) to transform W303-1A. Rich medium was yeastextract-peptone (YEP), and selective synthetic complete medium(SC) lacked appropriate supplements to select for plasmids (28).

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TABLE 1. S. cerevisiae strains used in this study

Plasmids. To construct pMK52, which expresses Tos3 fused to green fluorescentprotein (GFP) from the native promoter, we used PCR to amplifya DNA fragment containing 600 bp of 5' sequence and the TOS3open reading frame. The PCR fragment was inserted into the BamHI/PstIsites of the centromeric, TRP1-marked vector pMK43. pMK43 containsthe yeast-enhanced GFP (yEGFP) fragment from pYGFP3 (6) andthe CYC1 terminator in the pRS414 backbone and was constructedby inserting the yEGFP fragment into the XhoI/ClaI site of pMK22,which contains the CYC1 terminator in pRS414. pOV22, carryingthe CSRE-lacZ reporter, has been described previously (35).

Assay of Snf1 kinase activity by phosphorylation of SAMS peptide. The procedure described previously (14) was followed. Briefly,protein extracts were prepared from yeast cells grown to mid-logphase and harvested by filtration. Cells were broken by beingvortexed with glass beads in buffer A (50 mM Tris-HCl, pH 7.5,50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM dithiothreitol,0.1 mM phenylmethylsulfonyl fluoride, 10% [vol/vol] glycerol).After brief centrifugation, Snf1 was partially purified by DEAE-Sepharose(Amersham Biosciences) chromatography. Snf1 was eluted fromthe column with buffer A containing 0.2 M NaCl. Peak fractions(2 ml) were pooled and assayed for phosphorylation of the SAMSpeptide (HMRSAMSGLHLVKRR) (8) in the presence of [ ${gamma}$ -³²P]ATP (Perkin-Elmer;specific activity, $~$ 300,000 cpm/nmol) in reaction buffer (50mM HEPES, pH 7.5, 5 mM MgCl₂, 1 mM EDTA, 0.2 mM ATP, 10% [vol/vol]glycerol) containing 0.2 mM SAMS peptide. Kinase activity wasexpressed as nmol of phosphate incorporated into SAMS peptideper min per mg protein (8).

Immunoblot analysis. Protein extracts were prepared as described above. Proteinswere separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (8% polyacrylamide) and analyzed by immunoblottingusing polyclonal anti-Snf1 (5) or peroxidase antiperoxidase(Sigma-Aldrich). Antibodies were detected by enhanced chemiluminescencewith ECL Plus reagents (Amersham Biosciences).

Microscopy. Nuclei were stained by addition of 4',6'-diamidino-2-phenylindole(DAPI) (0.8 µg/ml) for 5 min. Cells were harvested bybrief centrifugation, resuspended in medium, and viewed usinga Nikon Eclipse E800 fluorescent microscope. Images were takenwith an Orca100 (Hamamatsu) camera, using Open Lab (Improvision)software, and processed in Adobe Photoshop 5.5.

RESULTS

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Results

Tos3 affects growth on nonfermentable carbon sources. The tos3 ${Delta}$ mutation conferred no significant defect in growthon plates containing 2% glycerol plus 3% ethanol (glycerol-ethanol)as the carbon source, regardless of whether cells expressedall three forms of Snf1 protein kinase or only Snf1-Sip1, Snf1-Sip2,or Snf1-Gal83 (14, 16) (Fig. 1A). However, comparison of thegrowth kinetics of tos3 ${Delta}$ mutant and wild-type cultures in richmedium (YEP) containing glycerol-ethanol at 30°C showeda difference in growth rate during mid-log phase. The doublingtime for wild-type cultures (W303-1A) was 2.8 h, whereas thatfor tos3 ${Delta}$ mutant cultures was significantly longer, 3.5 h forMCY5120 and 3.6 h for MCY5114 (Fig. 1B and data not shown).The final cell density was also much lower for tos3 ${Delta}$ mutant culturesthan for wild-type cultures (after 72 h of cultivation, opticaldensity at 600 nm was 5.9 for MCY5114 and 8.1 for W303-1A).In contrast, cultures of tos3 ${Delta}$ mutant and wild-type cells showedidentical growth kinetics in YEP-2% glucose or YEP-2% raffinoseduring exponential phase and through entry into stationary phase(data not shown). After incubation in YEP-2% glucose for 10days, tos3 ${Delta}$ mutant and wild-type cultures showed no differencein cell viability, whereas viability of snf1 ${Delta}$ mutant cultureswas more than 10-fold lower (data not shown). The tos3 ${Delta}$ and wild-typecultures also showed the same exponential growth rate in YEP-2%glucose containing 1 M sorbitol (doubling time, 1.2 h).

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FIG. 1. Growth of wild-type and tos3 ${Delta}$ mutant cultures. (A) Strains were W303-1A (wild type, WT), MCY4908, MCY5114, MCY4100, and MCY4975. Cultures were grown in YEP-2% glucose to mid-log phase and spotted in twofold serial dilutions on YEP-2% glucose and YEP-2% glycerol-3% ethanol solid medium. Plates were incubated at 30°C and photographed after 4 days. (B) Strains were wild type (WT) W303-1A (circles) and tos3 ${Delta}$ mutant MCY5114 (triangles). Cells were grown in 500-ml baffled flasks containing 100 ml of YEP-2% glycerol-3% ethanol. An agitation speed of 220 rpm was maintained throughout the cultivation at 30°C. Similar results were obtained with another tos3 ${Delta}$ strain, MCY5120. OD₆₀₀, optical density at 600 nm.

Tos3 affects Snf1 catalytic activity in cells grown on glycerol-ethanol. To assess the role of Tos3 in activating Snf1 protein kinase,we assayed Snf1 catalytic activity in cells grown to exponentialphase in glycerol-ethanol (Fig. 2A). For comparison, we alsoassayed cells grown in 2% glucose and shifted to 0.05% glucosefor 30 min (Fig. 2B). Cells were in all cases collected by filtration.Snf1 protein kinase was partially purified, and catalytic activitywas assayed by phosphorylation of the SAMS synthetic peptidesubstrate (8). Immunoblot analysis of assayed fractions wascarried out to determine levels of Snf1 protein (Fig. 2C).

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FIG. 2. Assays of Snf1 catalytic activity. (A and B) Cultures of wild-type (WT) and mutant strains of the indicated genotypes either were grown to mid-log phase in YEP-2% glycerol-3% ethanol (A) or were grown to mid-log phase in YEP-2% glucose and were then shifted to 0.05% glucose for 30 min (B). Snf1 catalytic activity was assayed by phosphorylation of the SAMS peptide substrate. Extracts were prepared from two independent cultures, and in each case at least three assays were carried out at different dilutions. Values are averages of results of at least six assays from the two experiments, and standard errors are shown. Cultures of two independent segregants of the same genotype were used for the experiment shown in panel A to ensure that the results reflect the indicated genotype. Representative strains are listed in Table 1. Results in panel B are in close agreement with previously reported experiments (14). (C) Immunoblot analysis of Snf1 protein in assayed fractions from panel A. Previous work showed that Snf1 protein levels were not affected by the tos3 ${Delta}$ or pak1 ${Delta}$ mutationunder the growth conditions used for panel B (14). (D) Strains MCY5510 and MCY5538, expressing Sip2 tagged with the TAP epitope at its C terminus, were grown to mid-log phase in YEP-2% glucose (Glu) or YEP-2% glycerol-3% ethanol (GE). Protein extracts were prepared, and immunoblot analysis was carried out. Anti-Snf1 antibody detected both TAP-tagged Sip2 and native Snf1 protein, which served as the loading control.

During growth in glycerol-ethanol, the tos3 ${Delta}$ mutation reducedSnf1 catalytic activity twofold (Fig. 2A) but did not reducelevels of Snf1 protein (Fig. 2C). In the experiment shown here,the tos3 ${Delta}$ mutant fraction appears to contain slightly more Snf1than the wild-type fraction (Fig. 2C), but this was not confirmedby immunoblot analysis of a different pair of fractions witha series of dilutions of the protein samples (data not shown).In contrast, Tos3 had little or no role during the responseto abrupt glucose depletion (Fig. 2B), in accord with previousresults (14). Pak1 was previously shown to be the most importantof the three Snf1 protein kinase kinases for activating Snf1upon glucose depletion (14) (Fig. 2B). Pak1 similarly playsan important role during growth in glycerol-ethanol; pak1 ${Delta}$ mutantcells showed much-reduced Snf1 catalytic activity (Fig. 2A)despite normal levels of Snf1 protein (Fig. 2C).

We then tested strains expressing only one of the three formsof Snf1 protein kinase. During growth in glycerol-ethanol, tos3 ${Delta}$ reduced Snf1 activity in the sip1 ${Delta}$ gal83 ${Delta}$ and sip1 ${Delta}$ sip2 ${Delta}$ mutantstrains, which express only Snf1-Sip2 and Snf1-Gal83, respectively(Fig. 2A). The sip2 ${Delta}$ gal83 ${Delta}$ strain, which expresses only Snf1-Sip1,had very low activity (about 10% that of wild type; data notshown) and was not further examined. In contrast, tos3 ${Delta}$ had noeffect during a shift to 0.05% glucose (Fig. 2B).

During growth in glycerol-ethanol, Snf1 activity in both sip1 ${Delta}$ sip2 ${Delta}$ and sip1 ${Delta}$ gal83 ${Delta}$ strains was half that of wild-type cellsexpressing all three forms of Snf1 protein kinase, whereas aftera shift to 0.05% glucose, activity in the sip1 ${Delta}$ sip2 ${Delta}$ mutant wasclose to that of wild type and activity in the sip1 ${Delta}$ gal83 ${Delta}$ mutantwas much reduced (compare Fig. 2A and B). These findings suggestthat Snf1-Sip2 is responsible for a greater fraction of Snf1activity, compared to Snf1-Gal83, during growth in glycerol-ethanolthan after a shift to low glucose. Correspondingly, expressionof Sip2, TAP tagged at its C terminus, from the genomic locuswas much higher during growth in glycerol-ethanol than duringgrowth in glucose (Fig. 2D). This finding is in accord withmicroarray studies showing that SIP2 RNA levels are regulatedby carbon source (10) and with immunoblot analysis of GFP-taggedß subunits expressed from centromeric plasmids, whichshowed that Sip2 levels were higher in glycerol-grown cellsthan in glucose-grown cells and became comparable to those ofGal83 (36). Levels of Sip2-TAP were, however, similar in wild-typeand tos3 ${Delta}$ mutant cells grown in either glucose or glycerol-ethanol(Fig. 2D).

Consistent with these findings that Tos3 has a role in activatingSnf1 during growth in glycerol-ethanol, we detected Snf1 catalyticactivity in cells expressing Tos3 alone (pak1 ${Delta}$ elm1 ${Delta}$ cells; strainMCY4970). The activity was low (0.69 ± 0.06 nmol/min/mg)but significantly higher than that found when cells were grownin 2% glucose (0.28 ± 0.04 nmol/min/mg). Another pak1 ${Delta}$ elm1 ${Delta}$ strain (MCY5121) showed similar Snf1 activity when grownin glycerol-ethanol (0.53 ± 0.06 nmol/min/mg).

Tos3 affects activation of the CSRE. To address the role of Tos3 in Snf1-dependent gene expression,we examined the effects of the tos3 ${Delta}$ mutation on activation ofthe Snf1-dependent carbon source-responsive element (CSRE) ofgluconeogenic genes, which binds the activators Cat8 and Sip4(26, 35). Expression of gluconeogenic genes is essential forgrowth on a nonfermentable carbon source. Snf1-Gal83 is themost important form of the kinase for activation of the CSRE(38; data not shown), so we assayed expression of a CSRE-lacZpromoter fusion in sip1 ${Delta}$ sip2 ${Delta}$ cells, which express only Snf1-Gal83.When cells were shifted from 2% glucose to glycerol-ethanolfor 5.5 h, the tos3 ${Delta}$ mutation reduced expression of ß-galactosidase(Fig. 3A); however, when cells were shifted to 0.05% glucose,the reporter was activated much less strongly, and tos3 ${Delta}$ hadno effect (Fig. 3B). In contrast, the pak1 ${Delta}$ mutation reducedactivation in both cases. Thus, the effects of tos3 ${Delta}$ on the functionof a Snf1-dependent promoter element in vivo correlate wellwith its effects on activation of Snf1 catalytic activity.

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FIG. 3. Effect of tos3 ${Delta}$ on CSRE-lacZ expression. Cells of the indicated genotype were transformed with pOV22, carrying the CSRE-lacZ reporter (35). Transformants were grown selectively in SC-2% glucose to mid-log phase and then were shifted to SC-2% glycerol-3% ethanol (A) or to SC-0.05% glucose (B) for 5.5 h. ß-Galactosidase activity was assayed in permeabilized cells and is expressed in Miller units. Values are averages of assays of four transformants, and standard errors are shown. Control transformants carrying the parental vector gave negligible ß-galactosidase activity under both conditions (not shown). WT, wild type.

Expression and localization of Tos3 in glycerol-ethanol. To assess the expression of Tos3, we introduced a C-terminalTAP tag at the genomic TOS3 locus in strain MCY5533. Cultureswere grown in YEP containing glucose or glycerol-ethanol, andexpression of Tos3-TAP was examined by immunoblot analysis.No differences in protein levels or mobility were observed (Fig.4A). These findings are consistent with evidence that TOS3 RNAis not strongly regulated in response to carbon source (10).

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FIG. 4. Expression and localization of Tos3. (A) Strain MCY5533, expressing Tos3 tagged with the TAP epitope at its C terminus, was grown to mid-log phase in YEP-2% glucose (Glu) or YEP-2% glycerol-3% ethanol (GE). Protein extracts were prepared and subjected to immunoblot analysis. (B) Wild-type strain W303-1A was transformed with centromeric plasmid pMK52, expressing Tos3-GFP from the native promoter. Cells were grown to mid-log phase in selective SC-2% glucose and were shifted to SC-0.05% glucose for 30 min. Cells were also grown in SC-2% glycerol-3% ethanol. GFP fluorescence and DAPI staining are shown.

The subcellular localization of Tos3 has not been reported previously.To determine its localization, we expressed Tos3-GFP from itsnative promoter on a centromeric plasmid. This fusion proteinwas functional, as tos3 ${Delta}$ pak1 ${Delta}$ elm1 ${Delta}$ triple mutant cells expressingTos3-GFP were able to grow on raffinose. The localization ofTos3-GFP was examined in wild-type cells during growth in 2%glucose, after a 30-min shift to 0.05% glucose, and during growthin glycerol-ethanol. Tos3-GFP was cytosolic and excluded fromthe nucleus under all conditions (Fig. 4B).

DISCUSSION

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Discussion

We show that, when cells are grown on glycerol-ethanol, thetos3 ${Delta}$ mutation reduces growth rate, Snf1 catalytic activity,and activation of the Snf1-dependent CSRE of gluconeogenic genes.This is the first demonstration of any phenotype for tos3 ${Delta}$ , beyondthe initial finding that the tos3 ${Delta}$ pak1 ${Delta}$ elm1 ${Delta}$ triple mutant exhibiteda Snf^– phenotype not found in any of the double mutants.These results indicate that Tos3 contributes to activation ofSnf1 protein kinase during growth on nonfermentable carbon sources.In contrast, the tos3 ${Delta}$ mutation did not significantly affectSnf1 catalytic activity or CSRE function during abrupt glucosedepletion, indicating that Tos3 has a much more substantialrole during growth on nonfermentable carbon sources than duringacute carbon stress. These findings lend support to the ideathat the three Snf1 protein kinase kinases make different contributionsto cellular regulation under different growth conditions.

Nonetheless, the defects that we have detected are modest, andeven during growth on glycerol-ethanol, Pak1 affects Snf1 activitymore profoundly than does Tos3. The abundance of Tos3 was notestimated in the yeast GFP fusion localization database (11),because Tos3-GFP was not detected, and it is possible that Tos3is simply less abundant than Pak1. We also consider it possible,however, that the primary function of Tos3 involves activationof Snf1 protein kinase in response to some other environmentalcondition.

In addition, Tos3 may have some role in the cell that is unrelatedto Snf1. For example, Tos3 may phosphorylate other protein kinasesor other components of a different regulatory pathway. Anotherof the Snf1-activating kinases, Elm1, has roles in cell cycleprogression, cell morphology, and filamentous invasive growth(2, 3, 7, 30, 31) that are apparently unrelated to Snf1. Wealso note that a mammalian ortholog of Tos3, the LKB1 tumorsuppressor kinase, activates not only AMPK (13, 16, 40) butalso a dozen other AMPK-like protein kinases (21). Thus, theexistence of three Snf1 protein kinase kinases may reflect diversecellular functions that go far beyond the activation of Snf1.

ACKNOWLEDGMENTS

We thank K. Hedbacker for assistance with microscopy and H.Wiatrowski and K. Hedbacker for strains.

This work was supported by NIH grant GM34095 to M.C. and inpart by a KOSEF fellowship to M.-D.K.

FOOTNOTES

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

REFERENCES

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