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FEMS Yeast Research 5 (2005) 767–776

Deficiency of Pkc1 activity affects glycerol metabolism inSaccharomyces cerevisiae

Katia N. Gomes a, Suzy M.A.C. Freitas a, Thiago M. Pais a, Juliana L.R. Fietto a,Antonio H. Totola a, Rosa M.E. Arantes b, Antonio Martins c, Candida Lucas c,

Dorit Schuller c, Margarida Casal c, Ieso M. Castro a, Luciano G. Fietto a,Rogelio L. Brandao a,*

a Laboratorio de Biologia Celular e Molecular, Nucleo de Pesquisas em Ciencias Biologicas, Deparatmento de Farmacia, Escola de Farmacia,

Universidade Federal de Ouro Preto, Campus do Morro do Cruzeiro, 35.400-000 Ouro Preto, MG, Brazilb Departamento de Patologia Geral, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Campus da Pampulha,

Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazilc Centro de Biologia (CB-UM), Departamento de Biologia, Universidade do Minho, 4710-057 Braga, Portugal

Received 28 July 2004; received in revised form 4 January 2005; accepted 17 January 2005

First published online 14 March 2005

Abstract

Protein kinase C is apparently involved in the control of many cellular systems: the cell wall integrity pathway, the synthesis ofribosomes, the appropriated reallocation of transcription factors under specific stress conditions and also the regulation of N-gly-cosylation activity. All these observations suggest the existence of additional targets not yet identified. In the context of the controlof carbon metabolism, previous data had demonstrated that Pkc1p might play a central role in the control of cellular growth andmetabolism in yeast. In particular, it has been suggested that it might be involved in the derepression of genes under glucose-repres-sion by driving an appropriated subcellular localization of transcriptional factors, such as Mig1p. In this work, we show that apkc1D mutant is unable to grow on glycerol because it cannot perform the derepression of the GUT1 gene that encodes glycerolkinase. Additionally, active transport is also partially affected. Using this phenotype, we were able to isolate a new pkc1D revertant.We also isolated two transformants identified as the nuclear exportin Msn5 and the histone deacetylase Hos2 extragenic suppressorsof this mutation. Based on these results, we postulate that Pkc1p may be involved in the control of the cellular localization and/orregulation of the activity of nuclear proteins implicated in gene expression.� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Protein kinase C; Glucose repression; Glycerol transport; Glycerol metabolism

1. Introduction

Glycerol, besides being the compatible solute thatSaccharomyces cerevisiae accumulates under osmotic/salt stress, is also a key metabolite, its pathway playinga central role in regulation of processes, such as redox

1567-1356/$22.00 � 2005 Federation of European Microbiological Societies

doi:10.1016/j.femsyr.2005.01.003

* Corresponding author. Fax: +55 31 3559 1680.E-mail address: rlbrand@nupeb.ufop.br (R.L. Brandao).

balance, Pi availability and lipid synthesis. Glycerolcan also be used as carbon source and is transportedsimultaneously by the constitutively expressed Fps1pchannel [1–3] and by a proton symporter repressed byglucose and inducible by growth on non-fermentablecarbon sources [4,5]. Furthermore, glycerol is catabo-lized in S. cerevisiae through two subsequent enzymaticsteps. The first leads to the production of glycerol3-P, an important intermediate for lipid synthesis, by

. Published by Elsevier B.V. All rights reserved.

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glycerol kinase encoded by GUT1 [6,7]. The second met-abolic step connects glycerol consumption to glycolysisthrough the production of dihydroxyacetone-P by aFAD-dependent glycerol 3-P dehydrogenase encodedby GUT2 [8]. This enzyme is localized in the outer mem-brane of mitochondria and plays, together with the cyto-solic NADH-dependent glycerol 3-P dehydrogenase(Gpd1p, Gpd2p), a determinant role on the redox shut-tle between mitochondria and cytoplasm [9].

Consistent with these multiple roles, a major signal-ling pathway has been named the High-OsmolarityGlycerol (HOG) pathway, modulating glycerol synthesisthrough the transcriptional control of the GPD genes.This plays an important role in vital responses to osmo-tic, salt or temperature stress [10]. Furthermore, it inter-plays with other signalling pathways involved in cellshaping, polarization, mating and integrity, in particularthe PKC-mediated MAP Kinase pathway [11,12].

Both GUT1 and GUT2 have long been known to beunder glucose repression [13,8]. GUT1 repression isindependent of Mig1p and occurs through Opi1p, arepressor involved in inositol metabolism. The Adr1pand Ino2/Ino4p are responsible for more than 90% ofGUT1 expression under derepression conditions [13].Recently, it has been demonstrated that chromatin bind-ing of Adr1p is controlled by Snf1 protein kinase [14],whereas Opi1p activity is controlled by phosphorylationin a PKC-dependent mechanism [15]. These data are inaccordance with the existence of a complex net acting inthe control of glycerol metabolism in yeast, consistentwith the multiple roles mentioned above.

The role of Pkc1p in the control of carbon metabo-lism seems to be rather broad, since a pkc1D mutantdisplays defects in fermentation initiation and derepres-sion of different enzymes upon glucose starvation [16].In addition, pkc1D has low respiratory capacity. Thismutant is also unable to use glycerol as sole carbonsource. In this context, inability of pkc1D to grow onglycerol may be due to a respiratory defect or to a linkbetween protein kinase C and the regulation of glycerolmetabolism. Moreover, pkc1D, when compared to thewild-type strain, shows poor or slow growth in mediacontaining galactose or raffinose, as well as a defectin derepression of invertase activity, i.e., SUC2 expres-sion upon transfer of cells from glucose to raffinose[17,16]. All these results suggest that Pkc1p activitycould be connected to the glucose repression mecha-nism in S. cerevisiae. It has been suggested that Pkc1pmay control the cellular localization of Mig1p tran-scriptional factor [16], independently of Snf1p activa-tion [18–20].

Besides the role in the control of carbon metabolism,the Pkc1p-mediated pathway is essential for the mainte-nance of cellular integrity by controlling the expressionof genes encoding enzymes involved in cell wall con-struction [21]. It seems to be connected to other signal

transduction pathways and/or cellular processes suchas the mating MAP kinase pathway [11], cell fusion[22], polarized growth [23], regulation of actin cytoskel-eton polarization [24,25], control of the morphogenesischeckpoint during the cell cycle [26], and several eventsthat affect the cell membrane [27]. Accordingly, the pro-tein kinase C-mediated pathway has been suggested tobe involved, in connection with calcineurin, a Ca2+/cal-modulin-dependent protein phosphatase, in the temper-ature-induced expression of FKS2, a gene encoding thecatalytic subunit of the 1,3-b-glucan synthase [28]. Thisenzyme is a dimmer containing Rho1p as regulatorysubunit. Rho1p also acts as a monomer upstream Pkc1pin the correspondent pathway [29].

Moreover, the protein kinase C-mediated MAPKinase pathway cascade regulates the expression of yetanother subset of yeast genes, targeting the formationof one of the two complex forms of RNA polymeraseII [30].

Concerning the carbon metabolism, in a previouswork [16], it had been shown that the pkc1D mutantcould not grow on glycerol. This could imply that thegenes involved consumption of this substrate are undercontrol of the protein kinase C-mediated pathway. Inorder to evaluate this possibility, the two first stepsneeded for glycerol catabolism, transport and phosphor-ylation by glycerol kinase, were studied. In this work, wedemonstrate that Pkc1p controls glycerol consumption,affecting the glycerol kinase activity by regulating theGUT1 gene expression via a MAP kinase-independentpathway. This reinforces the idea that eventually thereis a bifurcation at the level of Pkc1p with consequentalternative targets. We also demonstrate that the growthphenotype is not due to a defect in glycerol symporteractivity, which is affected but not abolished in the pkc1Dstrain.

Furthermore, a new mutant from the pkc1D strainpresenting a constitutive derepression phenotype hasbeen found, as well as two extragenic suppressors ofthis mutation that were identified as the nuclear expor-tin Msn5p and the histone deacetylase Hos2p. To-gether with previous data, the results presented heresupport the idea that Pkc1p could be involved in thecontrol of the cellular localization and/or regulationof the activity of nuclear proteins implicated in geneexpression.

2. Materials and methods

2.1. Strains and growth conditions

The S. cerevisiae strains used in this study were:W303-1A (Mat a ade2-1 his3-11,15 leu2-3,112 trp1-1ura3-1 can1-100 GAL mal SUC2), YSH813 (W303-1A + bck1D::LEU2), YSH850 (W303-1A + pkc1D::

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HIS3) and CLY3 (isogenic to W303 but gut1 [4]). TheEscherichia coli strain used was TOP10F 0 (F 0, mcrA, D(mrr-hadRMS – mcrBC) B 80D lacZ DM15, Dlacx74,deoR, rec D1, araD139, D (ara, leu), 7697, gal IU, galK,k�, rs2p, end D1, nupG). Yeast cells were grown at 30 �Cin YP medium (2% (w/v) peptone and 1% (w/v) yeast ex-tract) supplemented with variable concentrations of thecarbon sources glycerol, glucose, galactose, raffinose orfructose, and 1 M sorbitol as indicated. Growth wasmonitored by measuring OD at 600 nm or by a drop teston solid media. E. coli was grown at 37 �C in LB med-ium (1% (w/v) tryptone; 0.5% (w/v) yeast extract; 0.5%(w/v) NaCl; 1.5% (w/v) agar).

2.2. Determination of invertase and glycerol kinase

activities

Invertase activity was measured as described before[31] with the modification introduced by Celenza andCarlson [32], except that the assay was carried out atpH 5.1 and 37 �C. Glycerol kinase activity was deter-mined in cell-free extracts obtained in 50 mM imidazolebuffer without sorbitol. Measurement of specific activityof glycerol kinase was performed according to Castroand Loureiro-Dias [33] by following ADP formationand using 1 U ml�1 pyruvate kinase and lactate dehy-drogenase in the coupled reaction. The reaction wasstarted by the addition of 0.1 mM of glycerol. An extinc-tion coefficient of 6.22 (l mmol�1 cm�1) for NADH wasused for calculations. Enzyme specific activities were ex-pressed in relative units on total protein in cell-free ex-tracts. Protein was estimated by the method of Lowryet al. [34] using bovine serum albumin as standard.

2.3. Glycerol transport studies

Initial rates of glycerol uptake, as well as in/out accu-mulation ratios, were determined as previously de-scribed [5]. The intracellular volume value used todetermine intracellular glycerol molarity has been deter-mined by Lages et al. [35].

2.4. RNA isolation and Northern-blot analysis

For a shift from growth on glucose to growth onglycerol, yeast cells were grown in 20 ml YP with glucose(4% w/v) up to OD ± 0.8–1.2. The sample was split intwo and washed quickly by centrifugation with 1-M sor-bitol. One of the cellular pellets was used for RNAextraction (repressive state). The other was resuspendedin YPglycerol (3%, w/v) and glucose (0.05% w/v) plus 1-M sorbitol, rapidly mixed and incubated as before. After2 h, the sample was washed in the same way and the pel-let used for RNA extraction (derepressed state). Totalyeast RNA was isolated by the hot acid phenol method[36]; 15 lg of total RNA was separated on 1% (w/v)

agarose in 50 mM boric acid, 1 mM sodium citrate,5 mM NaOH, pH 7.5, containing 1% (w/v) formalde-hyde. Subsequently, RNA was blotted onto Hybond-Nmembranes in 10· SSC (1.5 M NaCl, 0.15 M sodium cit-rate, pH 7.0) and hybridized with gene-specific probes.These were obtained by PCR with the following primers:f5 0-AATAGTTATATGTTTCCC-3 0 and r5 0-GCTATT-TATGTTGTTATTGG-3 0 for GUT1 and f5 0-GCTGC-TTTGGTTATTGATAAC-3 0 and r5 0-GATAGTGGA-CCACTTTCGTCG-3 0 for ACT1 (constitutive endoge-nous control). Probes were radioactively labelled usingthe Rediprimee II labelling kit (Pharmacia). TheRNA levels were visualized by exposing the membraneto CL-X Posuree Film from Pierce.

2.5. Mutagenesis of yeast cells using UV radiation

Yeast cells were grown overnight (OD600nm ± 1), col-lected by centrifugation, washed and resuspended in4 ml 1-M sorbitol. Appropriated dilutions of this sus-pension (1/10; 1/100 and 1/1000) were plated and ex-posed to different UV doses (0, 2, 10 and 15 mJ).Immediately after, the cells were wrapped in aluminumfoil to avoid photo reactivation and incubated at 30 �Cfor 3–5 days. The mutants obtained in this process wereselected on YPglycerol plates plus 1-M sorbitol. Theywere subsequently transformed with a multi-copy yeastgenomic DNA library (YEP13 multi-copy vector,ATCC 37329), using the lithium acetate procedure[37]. The transformants were selected in SD-glucose + 1-M sorbitol [6] according to the auxotrophicmarks. In a second selection round, the colonies wereplated in medium YPglycerol + 1-M sorbitol. Rever-tants of the pkc1D mutant phenotype on glycerol wereselected and tested for invertase activity after growth(OD ± 1.5) on 4% (w/v) glucose and 2% (w/v) raffinose.

2.6. DNA manipulation

Plasmid DNAs were rescued from yeast transfor-mants [38] and amplified in E. coli TOP10F 0. Suitablerestriction fragments of the selected clone were sub-cloned and sequenced [39]. All reactions were carriedout using the ABI Prism BigDyee Terminator CycleSequencing Ready Reaction Kits and the ABIR 310DNA Analyser (Applied Biosystems, Foster City, CA,USA). After editing, sequences were compared in theSaccharomyces Genome Database to identify the over-expressed sequences.

To test mutations introduced through mutagenesis inthe genes MSN5 and HOS2, �0.3 lg of the genomicDNA extract from strain 335 was used as template instandard PCRs using specific primers. In each amplifica-tion, the products were analyzed by agarose gel electro-phoresis. The amplicons were sequenced and comparedwith the published MSN5 and HOS2 sequences. Three

Fig. 1. Glycerol kinase activity (Panel (a)) and GUT1 expression levels(Panel (b)) on S. cerevisiae wild-type and mutant strains. Cells weregrown in YPD supplemented with 1 M sorbitol, i.e. repressionconditions (R), and subsequently incubated in YPG with the sameamount of sorbitol for derepression (D), for 6 h (Panel (a)) and 2 h(Panel (b)). The expression was determined by Northern-blot analysis

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different amplicons for each gene were sequenced to en-sure that mutations were not introduced into DNA frag-ments by the PCR.

2.7. Subcellular localization of Mig1p by analysis of GFP

fluorescence

To study the subcellular localization of Mig1p, weused yeast cells (wild-type and mutant strains pkc1Dand 335) transformed with a plasmid containing thetransport domain of MIG1 fused to the GFP gene [38].Cells were grown on YP medium supplemented with4% (w/v) glucose until mid-exponential phase. Samples(1 ml) were harvested and washed by centrifugation(13,000 rpm in a microcentrifuge) with a cold solutionof sorbitol (1 M) plus glucose (4%, w/v). The remainingcell suspension was transferred to YP-raffinose (2%,w/v) medium for 1 h. The cells were harvested andwashed by centrifugation with a cold solution contain-ing sorbitol (1 M), plus glucose (2%, w/v) (repressedcells) or glycerol (3%, w/v) (derepressed cells). Finally,10–15 ll of these cell suspensions were mixed with 10–15 ll of agarose (1%, w/v) at 37 �C for microscopyobservations. Images were registered by using an Olym-pus BX51 TRF microscope with accessory apparatus forfluorescence. Nuclei were visualized using a DNA dyesolution containing 2% Hoechst 33342 (MolecularProbes, Eugene, OR, USA).

using a probe for the constitutive actin gene ACT1 as control.

Fig. 2. Radiolabelled glycerol accumulation ratios in S. cerevisiae cellscultivated on YPD supplemented with 1 M sorbitol and subsequentlyincubated for 6 h in YPG with the same amount of sorbitol forderepression: (h, wild-type W303-1A; n, bck1D;�, pkc1D;�, strain335 (new mutant isolated from UV radiation on pkc1D). Results aremedium values of at least three independent experiments.

3. Results

The strain deleted in the PKC1 gene is unable to growon non-fermentable carbon sources [16]. This growthdefect is not present in the bck1D (the first protein kinaseof the MAP kinase pathway) and wild-type (W303-1A)strains, indicating that it is not dependent on the down-stream components of the MAP kinase pathway [11]. Itis well known that glycerol kinase (encoded by GUT1) issubjected to glucose repression and required for glycerolmetabolism [4]. Therefore, we investigated whether glyc-erol kinase activity could be constitutively repressed inthe pkc1D mutant. In Fig. 1(a), we show that, contrarilyto the W303-1A (wild type) and bck1D strains, thepkc1D mutant presented a very low glycerol kinaseactivity after cells had been shifted from glucose to glyc-erol. Accordingly, Northern-blot analysis (Fig. 1(b)) evi-denced that the GUT1 gene was less efficientlyderepressed in the pkc1D mutant than in wild-type andbck1D strains.

Considering that a growth defect on glycerol mightbe caused not only by a defect in GUT1 expression butalso by a defect in active transport activity [4], we mea-sured glycerol uptake in the pkc1D mutant. This strainpresented a radiolabelled glycerol accumulation capac-ity approximately half of that of the wild-type strain

assayed in derepression conditions identical to the onesabove mentioned for Gut1p activity assays, i.e., growthin YPD with 1 M sorbitol and subsequently incubationin YPG in the presence of 1 M sorbitol (Fig. 2). Consis-

Fig. 3. Growth of S. cerevisiae strains on YP media containingdifferent carbon sources: (a) YPD; (b) YPD + 1 M sorbitol; (c)YPGlycerol + 1 M sorbitol; (d) YPRaffinose + 1 M sorbitol. Strain335 is a mutant isolated from UV radiation on pkc1D. Strains 342 and345 are transformants from strain 335.

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tently, uptake Vmax in the pkc1D mutant was288 ± 37 lmol h�1 g�1 (n = 3), while the estimated Vmax

in wild type was 392 ± 25 lmol h�1 g�1 (n = 3), a valuewithin the range of those determined previously in thesame strain [4,40]. The affinity of the carrier (Km) ineither strain remained within the predicted intervalaccording to previous statistical validation [40]:3.4 ± 0.6 mM (n = 4) and 3.5 ± 1.3 mM (n = 6) in wildtype and pkc1D, respectively [4,5,40]. Additionally, thebck1D strain was also assayed, presenting, consistentlywith the results above, unchanged transport ability(Vmax 329 ± 31 lmol h�1 g�1 (n = 3); Km 2.1 ± 0.5 mM(n = 3)) (Fig. 2).

Additionally, Vmax of glycerol uptake was determinedin the gut1D strain grown and subsequently incubatedunder identical conditions as mentioned above. Thiswas performed as a control for the pkc1Dmutant, takinginto consideration previous data that showed glycerolkinase to have an effect on the initial rate of glycerol up-take used for determination of transport kinetic con-stants [4,40]. The value obtained for Vmax was350 ± 41 lmol h�1 g�1 (n = 3), once more in the rangeof the wild type Vmax. This indicates that the reducedtransport velocity determined in the pkc1D mutant isnot an indirect consequence of the inability to expressthe Gut1p.

In order to get more information on the mechanismby which Pkc1p controls glycerol metabolism, a strategyinvolving the generation of new mutants was developed.The pkc1D mutant was exposed to UV irradiation, gen-erating 11 new mutants that recovered the capacity togrow on glycerol (not shown). However, they still didnot grow in the absence of sorbitol (not shown), suggest-ing that the new mutation probably did not occur in thedownstream components of the protein kinase C-medi-ated MAP kinase pathway. In this way, a new strategywas applied to obtain mutants able to revert the pkc1Ddefect of growth on non-fermentable carbon sources.One of the mutants was chosen, strain 335, for showingreversion of pkc1D phenotype on raffinose (slow growth)and/or glycerol (no growth) (Fig. 3).

In Fig. 1(a), we demonstrate that strain 335 presenteda higher glycerol kinase activity after the cells had beenshifted from glucose to glycerol. In the same way,Northern-blot analysis (Fig. 1(b)) revealed that theGUT1 gene was constitutively derepressed in this strainas compared to the wild-type and bck1D strains. Fur-thermore, mutant strain 335 presented uptake with aVmax of 324 (n = 2), in the same range as the valuesfound for the wild-type strain. Accordingly, accumula-tion ability was also not significantly different from thatmeasured in the wild type (Fig. 2).

On the other hand, in Fig. 3 it is shown that thisstrain grows on glycerol and therefore seems to haverecovered the wild type�s ability for respiratory growth.Consistently, Fig. 4 shows that, when grown in liquid

medium with glucose as carbon source, strain 335 pre-sents diauxic growth, the second phase being inhibitedby antimycin, known to block the respiratory chain.

Considering that invertase is one of the enzymesknown to be down-regulated by pkc1 deletion, theinvertase activity of this mutant was measured in cellsgrown on glucose and raffinose (Fig. 5) and comparedwith that of wild-type and pkc1D strains. As expected,in the wild type the classical pattern was observed: lowlevel of activity in glucose (or fructose-grown cells)and a high level in raffinose-grown cells, while in thepkc1D mutant, a low level of invertase activity was ob-served in both media. The mutant strain 335 presenteda very interesting phenotype, being apparently constitu-tively derepressed, since the invertase activity was highwith both carbon sources (Fig. 5).

Mig1p had previously been shown to remain in thenucleus in the pkc1D mutant, even under derepression[16]. For this reason, we decided to investigate the cellu-lar localization of Mig1p to see if the constitutive dere-pressive phenotype observed in the 335 mutant was dueto the fact that Mig1p would remain outside the nucleus.The results presented in Fig. 6 show Mig1p to be local-ized inside the nucleus in both strain 335 and pkc1D,either after repressive or derepressive growth conditions.This suggests that the derepressed phenotype of strain

Fig. 4. Growth of S. cerevisiae strains in YPD medium supplementedwith 1 M sorbitol in the absence (open symbols) and in the presence of2 lg ml�1 antimycin (closed symbols): (a) wild type; (b) pkc1; (c) strain335.

Fig. 5. Invertase activity in S. cerevisiae strains: wild type W303-1A,pkc1D, 335 mutant, 342 and 345 transformants. Cells were grown in (a)YPD or (b) YPraffinose with 1 M sorbitol.

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335, inferred from the results above, cannot be explainedby the control of the cellular localization of Mig1p.

Taking into account all these results, and consideringthe possibility that the 335 mutant could present a muta-tion in a gene encoding for a protein originally under thecontrol of Pkc1p, we complemented the strain 335 withtwo different yeast genomic libraries. Our selection strat-egy was based on the recovery of the inability to growon glycerol. We were able to recover five transformantsusing the multi-copy library YEP13, all of which did notgrow in the absence of 1 M sorbitol (not shown). Wechose two of these strains, 342 and 345, for showing par-tial reversion phenotypes: these transformants cannotgrow on glycerol, like pkc1D (Fig. 3); nevertheless theycan still grow on raffinose, although with certain diffi-culty. Moreover, when grown in liquid medium withglucose as carbon source, they did not present diauxicgrowth (data not shown). When the invertase activitywas measured, these strains presented an intermediaryactivity (Fig. 5) compared to those observed in thepkc1D mutant and strain 335.

The insert isolated from strain 342 had a length ofaround 4600 base pairs, containing an open readingframe of 3675 bp corresponding to the MSN5 gene se-quence, which codes for a nuclear exportin. On the otherhand, the DNA fragment extracted from strain 345 was2300 bp, and contained the complete sequence of theHOS2 gene, encoding a Class I histone deacetylase.These two genes were isolated by PCR from strain 335and further sequenced, showing no mutation. We there-fore consider that they apparently encode putativeextragenic suppressors of the derepression phenotypeof strain 335.

4. Discussion

Pkc1p seems to be involved in many important cellu-lar functions beyond controlling cell wall synthesis

Fig. 6. Subcellular localization of Mig1p in S. cerevisiae wild type,pkc1D and 335 mutant strains. Cells were transformed with a plasmidcontaining the MIG1 transport domain fused with GFP gene. Aftergrowth on YPD (with sorbitol), cells were transferred to YP-raffinosefor 1 h. The white arrowheads indicate the position of the nuclearregion observed in the yeast cells.

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through the protein kinase C-mediated MAP kinasepathway. There are evidences indicating the participa-tion of Pkc1p in the control of ribosomal synthesis inthe appropriated reallocation of transcriptional factorsunder specific stress conditions [41], and also in the reg-ulation of N-glycosylation activity [42]. Pkc1p seems tobe essential to the glucose-induced activation of theplasma membrane ATPase [43] and also for the dere-pression mechanism of glucose-repressible genes[17,16]. Together, these data support the idea that Pkc1pis involved in many important functions in yeast cells.The present work goes further into the mechanismsunderlying the involvement of Pkc1p in the control ofdifferent glucose-mediated effects.

Saccharomyces cerevisiae can utilize glycerol as solecarbon and energy source, whereas the pkc1D mutantis unable to grow on this substrate [17,16]. Glycerol up-take can be performed through the Fps1p channel[44,45] or by a glycerol/proton symport [4,5]. Once in-side the cell, glycerol is first converted into glycerol-3-phosphate by the enzyme glycerol kinase (Gut1p), andthis is oxidized to dihydroxyacetone phosphate by themitochondrial enzyme glycerol-3-phosphate dehydroge-nase (Gut2p). Cells presenting deletions in both theGUT1 and GUT2 genes are unable to grow in mediacontaining glycerol as carbon source, suggesting thatthis is the unique pathway operating for the glycerolcatabolism in S. cerevisiae [7].

In this work, we demonstrated that a pkc1D mutantcould not grow on glycerol because the transcriptionof the GUT1 gene and consequently glycerol kinaseactivity are abolished. Furthermore, we showed that ac-tive transport of glycerol in this strain is affected, sinceits accumulation capacity is considerably reduced inrelation to that of the wild type, concomitantly with a

reduction of �30% in the corresponding Vmax. This ef-fect appears to be independent of the downstream com-ponents of the protein kinase C-mediated MAP kinasepathway, since in the bck1D mutant both glycerol kinaseactivity as well as GUT1 expression and transport activ-ity present the same pattern as observed in the wild type.These results seem to confirm the existence of a bifurca-tion at the level of Pkc1p in this pathway. Moreover, thepkc1D mutant shows low invertase activity, regardless ofthe carbon source used for cultivation.

For this reason, we decided to try to identify newputative components of a pathway under direct controlof Pkc1p and involved in the derepression of glucose-repressed genes. The isolated mutant (strain 335) re-verted the pkc1D phenotype: it recovered the ability togrow on glycerol and presented a constitutive derepres-sion of glycerol kinase as well as of invertase activity.Concomitantly, mutant 335 was still able to grow fol-lowing glucose exhaustion, supposedly consuming respi-rable substrates like ethanol produced during glucoseconsumption.

Pkc1p seems to be responsible for the phosphoryla-tion of Opi1p that is a negative regulator of GUT1

expression [13,15]. It is still not clear how the phosphor-ylation of Opi1p affects its function. However, Pkc1p isinvolved in the reallocation of transcription factors[41,16], therefore, it seems reasonable to imagine thatphosphorylation of Opi1p might be important in thecontrol of its subcellular localization. Indeed, we havepreviously demonstrated that Pkc1p is important foran appropriate reallocation of the Mig1 transcriptionfactor [16], the action of which is essential for the glu-cose repression of several genes [46]. According to this,the constitutive repression exhibited by the pkc1D mu-tant could be at least partially explained by the perma-nent nuclear localization of Mig1p regardless of thecarbon source used. This finding agrees with the previ-ous suggestion of Pkc1p being essential for the controlof the subcellular localization of transcription factors[41]. In this context, Mig1p localization might be pre-dicted to be essentially extranuclear in the strain 335.However, our results demonstrate that Mig1p remainsin the nucleus in the mutant 335 under either repressiveor derepressive conditions, indicating that Mig1p is notthe agent responsible for the derepression observed inthis mutant. These results add a higher level of complex-ity to a hypothetical pathway where Pkc1p appears tohave a pivotal role in the derepression of different genes.

We were able to isolate two extragenic suppressors ofthe strain 335 phenotype: the b exportin Msn5, involvedin the traffic of transcriptional factors such as Mig1pfrom the nucleus to the cytoplasm [47,48], and thedeacetylase Hos2 that acts on chromatin structures en-abling the induction of some genes [49]. It has been sug-gested that Pkc1p may control the cellular localizationof the Mig1p transcriptional factor [16] independently

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of Snf1p activation [18–20]. Apparently, this transcrip-tional repressor contains a nuclear export signal that isphosphorylated by Snf1, provoking its recognition byMsn5p [47]. Msn5p was originally identified as a high-copy suppressor of the snf1 mutation [50,51]. It seemsto be a nuclear protein involved in different cellularprocesses such as carbon source utilization, calcium tol-erance, mating, and cyclin-specific functions [52]. More-over, Class I histone deacetylases are involved in thetranscriptional regulation of many genes by affectingthe chromatin organization [53]. Particularly, thedeacetylation of lysine residues in the H3 and H4 his-tone tails is required for interaction with Tup1p/Ssn6p,the co-repressor complex involved in the regulation ofmany glucose-repressed genes [54–57]. However, it hasbeen demonstrated that Hos2p and Rpd3p, both ClassI histone deacetylases, seem to have antagonistic ac-tions. Deacetylation triggered by Hos2p seems to be nec-essary for transcription induction of genes such as GALand INO1, while the Rpd3p activity is required forrepression. Apparently, differences between Hos2p andRpd3p in the dynamic chromatin binding, as well asthe additional deacetylation of H2A and H2B histonesby Rpd3, would be responsible for the different rolesin the gene regulation [49].

Although, these proteins are direct or indirectly in-volved in mechanisms controlling gene expression, theresults obtained in this work do not allow us to establisha conclusive relationship between Pkc1p activity andMsn5p or Hos2p. However, they still allow us to pro-pose a model mechanism in which Pkc1p would regulatea protein necessary for the activation and/or transloca-tion of nuclear proteins essential for the derepressionprocess. Thus, the mutations in the strain 335 wouldhave affected this protein, turning the derepression con-trol independent of Pkc1p. Following this model, theoverexpression of the MSN5 gene (strain 342) wouldpromote unspecific translocation of nuclear factors in-volved in derepression. On the other hand, the partialreversion of the original phenotype observed in strain335 by the overexpression of the HOS2 gene (strain345) is most probably a very different issue related tothe putative unspecific action of this histone deacetylaseon target sites of other similar enzymes like Rpd3,deacetylation of which is clearly involved in glucoserepression [55].

Since the proteins Mig1p and Hxk2p are directly in-volved in the glucose repression of genes like SUC2

[58], it is not possible to discard the hypothesis thatthe mutation in strain 335 occurred in the genes MIG1

and HXK2. However, it does not seem probable becausethere is no evidence indicating their participation eitherin the regulation of glycerol metabolism [59], or in thetranscriptional regulation of genes encoding for compo-nents of the respiratory apparatus [48]. Still, other tran-scription factors like Sko1 have been suggested to act

either as positive or negative regulator of transcriptionaccording to promoter specificity [12].

In conclusion, our findings do not allow us to pro-pose a precise mechanism by which Pkc1p participatesin the derepression process in yeast cells. However, wecan postulate that Pkc1p appears to be involved in thecontrol of the cellular localization and/or regulation ofthe activity of nuclear proteins implicated in glycerolmetabolism, in particular the glycerol symporter as wellas the glycerol kinase and the invertase. Further investi-gation will be needed to better understand the molecularbasis for these assumptions.

Acknowledgments

This work was financed by grants from FundacaoUniversidade Federal de Ouro Preto (fellowship toK.N.G.), from Fundacao de Amparo a Pesquisa do Es-tado de Minas Gerais – FAPEMIG (Brazil) ProcessCBS-1875/95 to R.L.B. and fellowships from ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico– CNPq (Brasil) Process 300998/89-9 to R.L.B. (re-search fellowship) and Process 301255/01-6 to L.G.F.(post-doctoral fellowship).

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