TURKEL, S. y ARIK, E. [2010] THE EFFECT Pho85 INVERTASE BIOSYNTHESIS AND GLUCOSE UPTAKE IN Saccharomyces cerevisiae.pdf

7/28/2019 TURKEL, S. y ARIK, E. [2010] THE EFFECT Pho85 INVERTASE BIOSYNTHESIS AND GLUCOSE UPTAKE IN Saccharomy

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Te efects o the Pho85 signaling pathway on invertase

biosynthesis and glucose uptake in Saccharomyces cerevisiae

Sezai RKEL*, Eli ARIK

Department o Biology, Faculty o Arts and Sciences, Uluda University, Bursa - URKEY

Received: 03.11.2008

Abstract: Glucose signaling controls a wide range o metabolic events in the yeast Saccharomyces cerevisiae. Glucosesensing and signaling processes require a variety o membrane-bound and cytoplasmic sensor proteins. Pho85p is acyclin-dependent protein kinase that controls diferent metabolic events upon binding to its cyclin partners in thecytoplasm o yeast cells. In this study, we investigated the roles o Pho85p in the glucose signaling pathways that controlinvertase biosynthesis and glucose uptake in yeast. Te biosynthesis o invertase enzyme rom the SUC2 gene is controlledby glucose repression and derepression mechanisms in S. cerevisiae. However, the results o this research indicated thatinvertase biosynthesis occurs at low levels in a deregulated manner in the pho85 mutant yeast strain. Furthermore, ourresults showed that the biosynthesis o invertase is not repressed when the pho85 mutant is exposed to high levels oglucose. Moreover, we ound that the glucose consumption rate o the pho85 mutant is at least 2-old lower than thato the wild-type yeast strain. Our results indicated that Pho85p unctions are essential or the regulated biosynthesis othe invertase enzyme rom the SUC2 gene and or the high levels o glucose uptake in S. cerevisiae.

Key words: Pho85, glucose sensing, glucose uptake, Saccharomyces cerevisiae, invertase

Saccharomyces cerevisiaeda Pho85 sinyal iletim yolann invertazbiyosentezi ve glukoz almna etkileri

zet: Glukoz sinyali Saccharomyces cerevisiaeda ok eitli metabolik olaylar kontrol eder. Glukozun alglanmas vesinyal iletimi ilemi iin hcre zar ve sitoplazmasnda bulunan eitli sensor proteinler gereklidir. Pho85p, sikline balbir protein kinaz olup ilgili siklin proteinlerine balanarak maya hcreleri sitoplazmasnda arkl metabolic olaylarkontrol eder. Bu almada Pho85pnin glukoz alm ve invertaz enzimi biyosentezini kontrol eden glukoz sinyal iletimiyolandaki ilevleri aratrld. S. cerevisiaeda invertaz enziminin SUC2 geninden biyosentezi glukoz basklanmas vebasknn kaldrlmas mekanizmas ile kontrol edilir. Bununla birlikte, bu aratrmada elde edilen sonular pho85

mutant maya hcrelerinde invertaz biyosentezinin dk seviyede ve dzensiz olarak meydana geldiini gstermektedir.Buna ek olarak, elde ettiimiz sonular pho85 mutantna yksek glukoz uygulandnda invertaz biyosentezininbasklanmadn gstermektedir. Ayrca, pho85 mutantnda glukoz tketim hznn yaban tip mayaya gre en az 2 katdaha dk olduunu belirledik. Sonularmz S. cerevisiaeda Pho85 ilevinin SUC2 geninden invertaz biyosentezininkontrol edilmesi ve yksek oranda glukoz alm iin gerekli olduunu gstermektedir.

Anahtar szckler: Pho85, glukoz alglama, glukoz alm, Saccharomyces cerevisiae, invertaz

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urk J Biol34 (2010) 103-108 BAKdoi:10.3906/biy-0811-4


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Te efects o thepho85 signaling pathway on invertase biosynthesis and glucose uptake in Saccharomyces cerevisiae

Introduction

Glucose is a preerred carbon and energy sourceor the yeast Saccharomyces cerevisiae (1). hepresence o glucose in the growth medium results inthe repression or activation o 30% o the genes in S.

cerevisiae (2,3). Depending on the extracellularglucose concentrations, either high ainity or lowainity sensors on the yeast cell membranes areactivated (4). Intracellular glucose sensors thatinteract with membrane-bound sensors transmit theglucose signals to the cytoplasmic regulatory proteins(4,5).

Pho85p is a cyclin-dependent kinase (CDK) thatinteracts with at least 10 dierent Pho85-dependentcyclins (Pcl) within S. cerevisiae cells (6). Pho85-Pclcomplexes control various metabolic pathways, such

as morphogenesis, and cell-cycle progression (6,7).Glycogen accumulation is one o the metabolic eventsthat Pho85 and its cyclins, Pcl6 and Pcl7, control inthe yeast S. cerevisiae (8).

Glucose is transported into yeast cells by the highainity or low ainity hexose transporters (9). hereare 20 hexose transporter-related genes (HXT) in S.cerevisiae. ranscriptions o certain HXTgenes arealso regulated by glucose signaling in yeast cells(9,10). Glucose sensing and signaling systems includemultiple actors (4,5). he invertase enzyme (E.C.

3.2.1.26) is encoded by the SUC2 gene (11). It isrequired or the utilization o sucrose and rainose ascarbon and energy sources or S. cerevisiae (11).Biosynthesis o the invertase is regulated by a glucoserepression mechanism at the transcriptional level (1,12). In the presence o high glucose levels (more than0.1% w/v) in the growth medium, transcription oSUC2 is repressed by the repressor proteins Mig1p,Ssn6p, up1p complex, and nucleosomes (13, 14).However, in the presence o low glucose (less than0.1%) or sucrose, transcription o SUC2 becomes

derepressed and is activated by transcription actors(15,16). Invertase is synthesized rom SUC2 mRNAand exported to the periplasmic area as an activeenzyme.

Previously, we have shown that the transcriptionoSUC2 and certain HXTgenes are regulated by thetranscriptional activator protein Gcr1p (15,17).ranscription o the HXT4 gene decreased about 35-

old in thegcr1 mutant (17). A signiicant decrease inthe low ainity glucose uptake system has beenreported or thegcr1 mutant yeast (18). In addition tothe deect in glucose uptake, SUC2 gene expression isalso deregulated in gcr1 mutant yeast cells. It wasshown that the expression o the SUC2 gene becomesinsensitive to glucose repression in this mutant (15).Gcr1p speciically binds to several sites on the SUC2,HXT2, and HXT4 genes promoter regions (15,17).Gcr1p is a phosphoprotein and interacts with thePho85p complex (19,20). Gcr1p requires Pho85p orits activation unctions (20). However, the exactphysiological role o Pho85p in the Gcr1p dependentmetabolic events such as glucose uptake and invertasebiosynthesis is not known. In this study, we analyzedthe involvement o Pho85p in glucose uptake andinvertase biosynthesis in S. cerevisiae.

Materials and Methods

Yeast strains and growth medium

he S. cerevisiae strains used in this research wereBY4741 (MAa, his31, leu20, met150, ura30)and Y02747 (MAa, his31, leu20, met150,ura30, YPL031c::kanMX4). Yeast strains BY4741and Y02747 are isogenic, except or the pho85mutation (21). hey were purchased rom theEUROSCARF yeast collection (University o

Frankfurt. Germany). The yeast strains were grown ina YPD medium (1% yeast extract, 2% peptone, 2%dextrose) with constant shaking (130 rev/min) at 30 Cin an incubator shaker. Glucose derepressed cells wereprepared as described previously (22).

Invertase assays

In order to compare the derepression rates o theinvertase biosynthesis in the wild-type and mutantstrains, yeast cells were irst grown in 50 mL o YPDmedium under the glucose repressed conditions untilthe early logarithmic stage (OD600: 0.70.8). Yeast cells

were then harvested and washed twice with an equalvolume o sterile distilled water. hey wereresuspended in 50 mL o resh YP medium thatcontained 0.05% glucose (w/v) or derepression oinvertase synthesis. he yeast cells were urtherincubated on a shaker (130 rev/min) at 30 C. hesamples were removed rom the cultures at speciictime intervals as indicated in Figure 1.


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In order to analyze the eects o Pho85p on therepression o invertase biosynthesis, yeast cells wereirst pre-cultured in 10 mL o YPGL medium (1%yeast extract, 2% peptone, 2% glycerol, 2% sodiumlactate) to the logarithmic stage. Glucose was added tothe portion o the yeast cultures at repressingconcentrations (2% w/v) and incubated or 2 h. At theend o the incubation period, yeast cells wereharvested and the invertase activities o the yeastcultures were determined as described by the glucoseoxidase peroxidase (GOD-POD) method (23,24).One unit o the invertase activity is equal to the

amount o enzyme that catalyzes the deliberation o 1mol glucose per minute per 100 mg dry weight oyeast cells (22).

Analysis o glucose consumption rates

In order to determine the glucose consumptionrates o the wild-type (BY4741) and the pho85mutant (Y02797), irst the yeast cells were cultivatedin 25 mL o YPD medium until the mid-logarithmicstage (OD600: 1.0). Yeast cells were harvested andwashed twice with an equal volume o sterile distilledwater. hen they were resuspended in 25 mL o a YP

medium that contained 0.1% glucose. Yeast cultureswere incubated on a shaker (130 rev/min) at 30 C.Samples (1 mL) were removed rom the cultures every20 min. Yeast cells in the samples were pelleted rapidlyby centriugation. he amounts o glucose in thegrowth medium were determined with a glucoseoxidase peroxidase assay, using 5-10 L o samples asdescribed (24). he glucose concentrations o the

growth medium were plotted versus the time points toobtain the glucose consumption patterns o the wild-type and the pho85 mutant yeast cells.

All experiments were done in triplicate andrepeated at least once under the same growth

conditions. Hence, the numbers that are given in thetables and igures are the mean values o at least 6independent experiments.

Results

Eects o Pho85p on the regulation o invertasebiosynthesis

In order to determine whether Pho85p is involvedin the regulated biosynthesis o invertase, invertaseactivities were measured in both the wild and the

pho85 mutant yeast strains. When the yeast cultureswere grown in a high glucose medium (2% w/v),invertase activities o the wild-type and the pho85mutant yeast cells were at their basal levels (35 units)(able 1). he invertase biosynthesis was activatedand yielded 1106 units in the wild-type yeast cellsonce they were transerred to the derepressed growthmedium. However, the invertase biosynthesis in thepho85 mutant was measured as 131 units in thederepressed growth medium (able 1). Invertasebiosynthesis in the pho85 mutant was approximately8-old lower than in the wild-type yeast cells,

indicating that the cyclin dependent kinase Pho85p isessential or the high level biosynthesis o invertaseenzyme in S. cerevisiae.

Moreover, we analyzed the involvement o Pho85pin the repression o invertase biosynthesis in

Time (min)

Invertase

activities

0 15 30 45 60 75 90 105 1200

100

200

300

400

500

600

700

800

900Wild Type

pho85 mutant

Figure 1. Derepression patterns o the invertase biosynthesis inthe wild-type and pho85 mutant yeast strains.

able 1. Invertase synthesized at low levels in pho85 mutantyeast.

Invertase activitiesa (SD)

Yeast Strains Repressed Derepressed

Wild Type 5 2 1106 110pho85 mutant 3 2 131 18

aInvertase activities are expressed as the amount o enzyme thatcatalyzes the deliberation o 1 mol glucose per minute per 100mg dry weight o yeast cells.(SD): Standard Deviations

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able 2. Invertase synthesis is not repressed ater long-termderepression in the pho85 mutant.

Invertase activitiesa (SD)

Yeast Strains Derepressed Repressed(YPGL) (YPGL to 2%

Glucose)

Wild Type 256 24 94 3pho85 mutant 123 8 124 1

aInvertase activities are expressed as the amount o enzyme thatcatalyzes the deliberation o 1 mol glucose per minute per 100mg dry weight o yeast cells.(SD): Standard Deviations

Time (min)

Glucoseconcentrations(mM)

0 25 50 75 100 125 1500.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

Wild Type

pho85 Mutant

Figure 2. Glucose consumption in the in the wild-type andpho85 mutant yeast strains.

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Te efects o the Pho85 signaling pathway on invertase biosynthesis and glucose uptake in Saccharomyces cerevisiae

derepressed yeast cells. hereore, the wild-type andthe pho85 mutant yeast strains were cultivated in aglycerol lactate medium, in which SUC2 geneexpression and invertase synthesis took place at amoderate level. When glucose was added to thealiquots o these yeast cultures, the invertase activitieso the wild-type yeast cells were repressed 3-oldwithin the 2 h o the incubation period in the presenceo high glucose levels in the growth medium.However, we ound that the invertase activitiesremained approximately at the same levels in thepresence o high glucose in the pho85 mutant yeaststrain (able 2). hese results suggest that Pho85pactivity is essential or the glucose signalingdependent regulation o invertase synthesis in S.cerevisiae.

Analysis o the derepression rate o invertasebiosynthesis

he biosynthesis o invertase is regulated byglucose signaling. In order to test whether there wasany change or luctuations during the derepressionstage, invertase activities in the pho85 mutant andin the wild-type yeast cells were determined in a time-dependent manner. Invertase biosynthesis wasderepressed rapidly in the wild-type yeast strains andreached its maximum levels (775 units) 90 min aterderepression (Figure 1). In contrast to the wild-typeyeast strain, derepression o invertase biosynthesis in

the pho85 mutant yeast strain occurred very slowly.Its invertase activity was 7-old lower than that o thewild-type yeast cells at the end o the incubation

period. his result clearly shows that although theinvertase biosynthesis was derepressed in the pho85mutant yeast strain in response to low glucose signalsit took place several times more slowly than that othe wild-type S. cerevisiae strain (Figure 1).

Eects o Pho85p on the glucose consumptionoS. cerevisiae

Glucose uptake and consumption is dependent onthe presence o an eicient and active glucosesignaling pathway in S. cerevisiae. Ater showing thatthe glucose signaling or the regulated biosynthesis othe invertase enzyme is deective in the pho85mutant yeast cells, we decided to analyze whether thePho85p unction is also essential or the normal levelo glucose consumption in the yeast cells. Glucoseconsumption was measured as the time-dependent

decrease in the glucose amount in the growthmedium o the yeast cells. he wild-type yeast cellsconsumed all o the glucose in the growth mediumwithin 90 min (Figure 2). he rate o glucoseconsumption was approximately 0.20.3mM/min/OD600 in the wild-type yeast cells underthese growth conditions. However, the glucoseconsumption rate in the pho85 mutant strain was 2-to 3-old lower than that o the wild-type yeast. hisconsumption rate was determined to be 0.1 to 0.15mM/min/OD600 in the pho85 mutant yeast strain(Figure 2). hese results indicated that the cyclin

dependent protein kinase Pho85p is required or thenormal level o glucose uptake in S. cerevisiae.


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1. Carlson M. Regulation o glucose utilization in yeast. Curr OpinGenet Dev 8: 560-564, 1998.

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Reerences

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S. RKEL, E. ARIK

Discussion and conclusions

Pho85p controls diverse metabolic events in S.cerevisiae (25). Our results indicated that the Pho85punctions are also essential or the glucose signalingthat results in the regulation o invertase biosynthesis

rom the SUC2 gene. Regulation o SUC2transcription by glucose signaling requires bothrepressors and activators (15,16). At high glucoselevels, the Mig1p complex, nucleosomes, and non-histone proteins Nhp6A/B repress the transcriptiono the SUC2 gene, which results in a very low level oinvertase activity in S. cerevisiae (13,26). SUC2 geneexpression is not ully repressed in pho85 mutantsat high glucose levels in the short term. It is knownthat the nucleosome positioning and the repressorprotein complex Mig1p-Ssn6p-up1ps complexbinding is required or the rapid and completerepression oSUC2 expression (16,27). Our results onthe eects o Pho85p on the biosynthesis o invertaseindicate that Pho85p targets at least one o the actorsthat participate in the repression o the SUC2 gene.One o the possibilities is that Pho85p may act on thechromatin binding and/or modiying complexes orrapid ormation o the repressed chromatin structureover the SUC2 promoter. As1p, which interacts withPho85p, unctions as a nucleosome assembly actor inS. cerevisiae (28-30). Hence, it is conceivable that thelow level activity o As1p in the pho85 mutant

might lead to the ineicient or slow ormation o arepressed chromatin structure over the SUC2promoter.

Lenburg and OShea (20) reported that Pho85p isrequired or the unction o Gcr1p and Spt7p in S.cerevisiae. Physical interactions between Pho85p andSpt7p were also shown (31). It is known that thetranscriptional regulation o SUC2 requires Gcr1pand SAGA histone acetyl transerase (HA) complex(15,32). Furthermore, it was shown that the lack ohistone acetyltranserase activity decreases the rate o

chromatin remodeling on certain promoters, whichthen leads to the signiicant drop in the rate o

transcriptional activation o targeted genes (33).Consistent with this observation, the lack o Pho85pactivity may lead to the slow remodeling o the SUC2promoter, resulting in the rather slow derepression oinvertase biosynthesis in the pho85 mutant.

We propose that the Pho85 dependentphosphorylation o Gcr1p in response to low glucosesignaling results in the rapid activation o invertasebiosynthesis rom SUC2 in the wild-type yeast cells.he same events may also occur in the transcriptionalregulation oHXTgenes. he expression o certainHXTgenes also depends on the Gcr1p and SAGAcomplex (17,34). Lack o Pho85p unctions in pho85mutant yeast might result in a less active Gcr1pcomplex that in turn leads to a low level o expressionoHXTgenes, which might result in the low level o

glucose consumption.Our results indicate that the Pho85p unctions are

essential or the intracellular glucose signaling thatresults in the regulated biosynthesis o invertase andthe high level o glucose uptake in S. cerevisiae.hereore, it appears that the slow growth phenotyperesults rom the ineicient glucose uptake andineicient glucose signaling in the pho85 mutantyeast strain.

Acknowledgmentshis research was supported by a research grant

rom the Scientiic and echnological ResearchCouncil o urkey (BAK, project no: 104307).

Corresponding author:

Sezai TRKEL

Department of Biology,

Faculty of Arts and Sciences,

Uluda University, 16059 Bursa, TurkeyE-mail: [email protected]


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TURKEL, S. y ARIK, E. [2010] THE EFFECT Pho85 INVERTASE BIOSYNTHESIS AND GLUCOSE UPTAKE IN Saccharomyces cerevisiae.pdf