Journal of Nuclear and Radiochemical Sciences, Vol. 8, No. 2, pp. 133-136, 2007

Protein Expression of Saccharomyces cerevisiae in Response to Uranium Exposure
F. Sakamoto,*, a T. Nankawa,a N. Kozai,a T. Fujii,b H. Iefuji,b A. J. Francis,c and T. Ohnukia,daAdvanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195, Japan
Environmental Research Division, National Research Institute of Brewing, Higashi Hiroshima, Hiroshima 7390046, Japanc
Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973, USAdEcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japanb

Received: January 31, 2007; In Final Form: March 26, 2007
Protein expression of Saccharomyces cerevisiae grown in the medium containing 238U(VI) and 233U(VI) wasexamined by two-dimensional gel electrophoresis. Saccharomyces cerevisiae of BY4743 was grown in yeast nitrogen base medium containing glucose and glycerol 2-phosphate and 238U of 0, 2.0, and 5.0 × 10 –4 M or 233U of 2.5 ×
10 –6 M (radioactivity was higher by 350 times than 2.0 × 10 –4 M 238U) and 5.0 × 10 –6 M for 112 h at 30 °C. The
growth of Saccharomyces cerevisiae was monitored by measuring OD600 at 112 h after the inoculation. Uranium
concentrations in the media also were measured by radiometry using a liquid scintillation counter. The growths ofthe yeast grown in the above media were in the following order: control > 2.5 × 10 –6 M 233U > 2.0 × 10 –4 M 238U >5.0 × 10 –6 M 233U > 5.0 × 10 –4 M 238U. This result indicated that not only radiological but also chemical effect of U
reduced the growth of the yeast. The concentrations of U in the medium containing 238U or 233U decreased, suggesting U accumulation by the yeast cells. The 2-D gel electrophoresis analysis showed the appearance of several
spots after exposure to 238U or to 233U but not in the control containing no uranium. These results show that theyeast cells exposed to U express several specific proteins.

1. Introduction
Yeast is known to accumulate U(VI).1,2 This suggests thatyeast may be utilized as bisorbents for removing U from U
mine tailings wastewater and from radioactive waste generatedat nuclear facilities. Since U is toxic for microorganisms, 3,4
yeast may be affected by exposure to U solution. Previously,
we showed that U(VI) retarded the growth of yeast in themedium containing uranium(VI).5 These results suggest thatthe exposure of yeast cells to U(VI) solution lead to a responseby change of metabolic pathways. However, to our knowledge,
little is known about response of yeast to U(VI) solution.
The use of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) have provided powerful tools for studying
protein expression during exposure of yeast cells to U(VI) solution. Two-D PAGE separates and allows visualization of mostproteins.6
In the present study, therefore, we examined the expressionof proteins in yeast exposed to U(VI). Protein expression wasstudied by 2-D gel electrophoresis, and expression pattern ofproteins was compared between U(VI) -exposed and nonexposed yeasts.
2. Materials and Methods
2.1. Strain and culture conditions. The yeast strain
Saccharomyces cerevisiae (S. cerevisiae) BY4743 (Funakoshi,Inc.) was grown in yeast nitrogen base (YNB) (with ammonium sulfate, without sulfates, without sodium chloride; QBIOgene, Inc.) medium. The YNB medium was prepared asfollows: 5.7 g of YNB, 20 g of glucose, 100 mg of glycerol 2phosphate disodium salt hydrate, 35 mg of DL-histidine, 30 mgof DL-lysine, 25 mg of uracil and 1 g of casamino acids. The
mixture was adjusted to 1 L with deionized water, and it was*Corresponding author. E-mail: sakamoto.fuminori@jaea.go.jp.
Fax: +81-29-282-5927.

autoclaved at 121 °C for 20 minutes. Uranium nitrate was dissolved in deionized water and the concentration was adjusted at
1.0 × 10 –1 M. We used 233U and 238U. Uranium-238 and U-233
emit α particles of 4.20 MeV (77%) and 4.15 MeV (23%) for
238
U and 4.82 MeV (84%), 4.78 MeV (13.2%), and 4.73 MeV(1.6%) for 233U. Half-lives of 238U and 233U are 4.5 × 109 y and1.6 × 105 y, respectively. The growth of the yeast was stopped
about 100 h under the culture condition. The effect of U for
the yeast was not observed when the concentration of U(VI)was 1 × 10 –4 M and the yeast did not grow when the concentration of U(VI) was 8 × 10 –4 M under the culture condition. Thus
the yeast was cultured in the YNB media containing 2 × 10 –4
and 5 × 10 –4 M U(VI) for 112 h at 30 °C. The initial amount ofyeast in the media was adjusted to 0.2 at the optical density at
600 nm (OD600).
The amount of the yeast in the liquid media was estimatedby measuring OD600 at 112 h after the inoculation. When OD600
of the media was beyond 2, it was diluted to 10 times withYNB liquid media. The U concentrations in the media weremeasured by radiometry using a liquid scintillation counter at112 h.
To compare proteins expressed by the cells exposed to U(VI)with that of control, yeast was cultured in the U(VI) freemedium for 112 h at 30 °C. The effect of α radiation on protein
expression by the yeast was examined using the medium containing 1.7 × 10 –10 and 3.4 × 10 –10 M of 233 U but not 238 U.
Radioactivity of 233U in the medium was higher by 350 times
than that of the 238U(VI) containing medium.
2.2. Protein extraction. “ReadyPrep Sequential Extraction
Kit” (Bio Rad, Inc.) was used for extraction of the yeast proteins. The yeast cells exposed to 238U of 2 × 10 –4 M, 233U of 2.5
× 10 –6 M, and U free media for 112 h were centrifuged 6000 ×g for 5 min. After removal of the supernatant, 20 mM TrisHCl (pH 7.4) was added to the cell pellet. The cell pellet wasresuspended, centrifuged again and the supernatant was discarded. The wet weight of the precipitation was measured.
Five hundreds milliliters of Reagent 1 (including the Kit) and 5© 2007 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 10/19/2007

134 J. Nucl. Radiochem. Sci., Vol. 8, No. 2, 2007

SakamotoµL of protease inhibitor cocktail were added to about 100 mg(wet weight) of the cells. The cells were lysed by the crushingmachine, “FastPrep FP 120” (Q-Biogene, Inc.). The supernatant was recovered, and its protein content was determined byusing the protein assay (Bio Rad, Inc.).
One hundredth of tributylphosphine was added to theReagent 2 (included in the extraction kit (Bio Rad, Inc.)).
About 5 µg of protein solution was adjusted at 125 µL by adding the Reagent 2 mixture. The solution was loaded by rehydration onto a 7-cm-long, pH 3 to 10 or 5 to 8 immobilized pH
gradient (IPG) strip (Bio Rad, Inc.) for 15 h at room temperature. Isoelectric focusing was carried out with PROTEAN IEF(Bio Rad, Inc.) for 15 min at 250 V (linear), 1 h at 8000 V (linear) and 1 h and 15 min at 8000 V (rapid) (total about 7500–9500 Vh). After focusing, the gel was reduced for 20 min atroom temperature in equilibration buffer 1 (50 mM Tris-HCl(pH 7.4), 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate(SDS), and 10 mg/mL dithiothreitol). After that, the gel wasalkylated for 20 min at room temperature in equilibration buffer 2 (50 mM Tris-HCl (pH 7.4), 6 M urea, 30% glycerol, 2%SDS, and 25 mg/mL iodoacetamide). The gel strips was transferred onto a 10–20% ReadyGel (Bio Rad, Inc.), and the second-dimension separation was carried out on the mini
PROTEAN system (Bio Rad, Inc.) at constant current (40 mA/gel). The gel was fixed and stained with the silver stain kit(Amersham Pharmacia, Inc.).

Chemical toxicity of U to bacteria was also suggested by
Suzuki and Banfield.4 Our results showed the effect of U on thecell growth depended on the concentration of U. The fractionof U accumulated on the cells was the same in the 238U concentrations of 2.0 × 10 –4 M and 5.0 × 10 –4 M.
Silver stained 2-D gel electrophoresis patterns of proteinsfrom cell lysate of the yeast grown in 2.0 × 10 – 4 M 233U are
shown in Figure 1. Proteins extracted by the Reagent 1 wereisoelectrically focused on the IPG gel of pH range from 3 to 10kDa(a)1003725103

3. Results and Discussion
The growth of yeast in YNB medium containing no U (control) and with U of 2.0 × 10 –4 M 238U, 5.0 × 10 –4 M 238U, 2.5 ×
10 –6 M 233U, and 5.0 × 10 –6 M 233 U for 112 h are shown in Table
1. Also shown in the table is the removal of U from solution in
the presence of cells. The growth of the yeast was affected by
the presence of U at concentrations 2.0 × 10 –4 M 238U and 5.0 ×10 – 4 M 238U. Extents of the growths, however, were smaller
than that grown in U free medium by 1.3 and 8, respectively. Itis indicated that the U concentration higher than 2.0 × 10 –4 M
affected the growth of yeast. In the case of 233U, the OD600 ofyeast grown in the medium containing 2.5 × 10 –6 M 233U wasslightly lower than U free medium and the OD 60 0 of yeast
grown in the medium containing 5.0 × 10 –6 M 233U was lowerby 1.4 than that in U free medium. It is indicated that higher
α-radioactivity of U affected more the growth of yeast. Theseresults strongly suggest that inhibition of growth was due toboth chemical and radiological effects.
More than 80% of U(VI) from solution were removed after
exposure of cells to the media containing U of 2.0 × 10 –4 M
238
U, 5.0 × 10 – 4 M 238U, 2.5 × 10 –6 M 233U, and 5.0 × 10 –6 M
233
U, indicating that most of the U in the media was accumulated by the cells. Soares et al.7 reported that S. cerevisiae
NCNY 1190 releases P when it is exposed to Cd, Cu, and Pb.
This release is attributed to U- or heavy-metal toxicity.746810pH(b)1005037252015105678pHFigure 1. Silver stained 2-D gel electrophoresis patterns of proteinsfrom cell lysate of the yeast grown in 2.0 × 10 – 4 M U containing
medium. Proteins extracted by the Reagent 1 were isoelectricallyfocused (a) on the IPG gel of pH range from 3 to 10, and (b) on theIPG gel of pH range from 5 to 8.

TABLE 1: The optical densities at 600 nm of yeast and fraction of U eliminated from medium at 112 h after the exposure tothe medium containing no U, 2.0 × 10 –4 M 238U, 5.0 × 10 –4 M 238U, 2.5 × 10 –6 M 233U, and 5.0 × 10 –6 M 233U
U concentration in the media

Initial optical densityof yeast

Optical density of yeast at 112 hafter exposure

no U (control)

0.2

2.79

–4

2.0 × 10 M

238

Fraction of U eliminated from medium
at 112 h after exposure

U

0.2

2.24

91.1

5.0 × 10 –4 M 238U

0.2

0.353

84.3

2.5 × 10 –6 M 233U

0.2

2.52

84.2

–6

0.21.9280.1

5.0 × 10 M

233

U

α-radioactivities in the medium are also shown. Initial optical density of yeast is adjusted to 0.2 at initial.

J. Nucl. Radiochem. Sci., Vol. 8, No. 2, 2007 135

Protein Expression of Saccharomyces cerevisiae in Response to(a)

kDa
100
50
3725
20
15
10567kDa

78pH(c)kDa
1005037252015106U 2.0 × 10 –4 M

U 5.0 × 10 –6 M

10050
37252015105238233(b)6U isotope and its
concentration in themedia8pH5TABLE 2: Isoelectric points (pH) and sizes of expressed
proteins of yeast cultured in the medium containing 238Uor 233U

78pH
Figure 2. Silver stained 2-D gel electrophoresis patterns of proteinsfrom the yeast grown in (a) U free medium, (b) 238U, or (c) 233U containing medium.

(Figure 1(a)). Many proteins were observed at nearly pH 6.
This indicated that the IPG gel of pH range from 5 to 8 gave
more appropriate pattern for the protein expression analysis ofthe yeast.
Silver stained 2-D gel electrophoresis patterns of proteinsfrom the yeast grown in the medium containing 238U or 233U are
shown in Figure 2, along with that of U free medium. In these,
the protein patterns depended on the culture conditions. The
circled protein spots in these patterns (Figure 2) showed thespots appeared after exposure to 238U or 233U, but not in control.
The protein spots corresponding to 2.0 × 10 –4 M 238U appearedat pH 5.9 and 19 kDa, pH 6.1 and 21 kDa, pH 6.8 and 35 kDa,

Character of expressed proteins
Isoelectric points/ pHSize/ kDa

5.9

19

6.1

21

6.8

35

6.9

35

7.4

16

5.9196.1217.2

20

7.4

16

It is noted that values of pH and protein size obtained were notabsolute ones.

pH 6.9 and 35 kDa, and pH 7.4 and 16 kDa. The protein spots
corresponding to 5.0 × 10 –6 M 233U appeared at pH 5.9 and 19kDa, pH 6.1 and 21 kDa, pH 7.2 and 20 kDa, and pH 7.4 and 16kDa.
Comparison of the protein spots corresponding to 2.0 × 10 –4
M 238U (Figure 2(b)) with those corresponding to 5.0 × 10 –6 M233
U (Figure 2(c)) showed that the proteins at pH 6.8 and 35kDa, and pH 6.9 and 35 kDa (shown by arrow head in Figure2(b)) were only expressed by 238U (Table 2). This result indicated that the proteins at pH 6.8 and 35 kDa, and pH 6.9 and 35kDa may be originated by response to chemical or radiologicaleffect of U. Similarly, the protein at pH 7.2 and 20 kDa (shownby arrow head in Figure 2(c)) was only expressed by exposure
to 233U, indicating that the protein may be originated by theresponse to α-irradiation. The proteins at pH 5.9 and 19 kDa,pH 6.1 and 21 kDa, and pH 7.4 and 16 kDa were expressed inboth conditions, indicating that the proteins were not specificproteins by U and α-irradiation. Additional studies are underway to distinguish the radiological effect on expression of specific proteins.
In this study, the proteins corresponding to exposure to 238Uor 233U was not characterized. Characterization of proteins byMALDI-TOF mass spectroscopy is planned.
4. Conclusion
Initial studies of protein expression of yeast exposed to U
was examined by two-dimensional (2-D) gel electrophoresis.
We confirmed that almost all proteins were observed at nearlypH 6. The 2-D gel electrophoresis patterns of the yeast proteins after exposure to 238U and 233U showed that proteins at pH
6.8 and 35 kDa, and pH 6.9 and 35 kDa were expressed in cells
exposed to 238U, but not to 233U. Protein at pH 7.2 and 20 kDa
was expressed in cells exposed to 233U, but not to 238U. Further
studies are underway to determine the effect of α-irradiationon the expression of proteins in yeast.
Acknowledgment. This research was supported in part bya Grant-in-Aid for Scientific Research B 18360459 from theMin ist r y of Education, Cult ure, Spor ts, Science a ndTechnology, Japan.
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