217 Biochimica et Biophysica Acta, 577 (1979) 217--220 © Elsevier/North-Holland Biomedical Press
BBA Report
BBA 31270 ISOLATION OF A THIAMINE-BINDING P R O T E I N F R O M SACCHAROMYCES CEREVISIAE
AKI0 IWASHIMA AND HIROSHI NISHIMURA Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamikyolzu, Kyoto (Japan) (Received November 6th, 1978) Key words: Thiamine-binding protein; (Saccharomyces cerevisiae)
Summary Thiamine-binding protein was isolated from Saccharomyces cerevisiae by successive procedures of cold osmotic shock treatment, DEAE-cellulose chromatography and ultrafiltration. The purified thiamine.binding protein was an electrophoretically homogeneous molecule which appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. No thiamine-binding protein was observed by disc gel electrophoresis in the shock fluid released from yeast ceils grown in the presence of I gM thiamine, indicating that the formation of this protein is regulated by exogenous thiamine as previously suggested.
Previous studies have shown that the transport of thiamine in Saccharomyces cerevisiae occurs by a carrier-mediated active process which is specific for thiamine [1,2]. It was also found that there exists a thiamine-binding protein in sonic extract of S. cerevisiae and it appeared to be repressed by exogenous thiamine with a concomitant decrease in thiamine uptake by yeast cells [3]. Purification of binding proteins from yeast cells,however, has been hampered by technical difficulties[4,5]. This paper describes the isolation of a thiamine-binding protein from S. cerevisiae. S. cerevisiae obtained as previously reported [2] was grown in 12 liters of thiamine-deficient Wickerham's minimal medium [6] at 30 ° C. They were harvested at the late exponential phase, and the cells were washed
218
twice with distilled water, then subjected to the cold osmotic shock treatment as follows. Washed yeast cells were suspended in 400 ml of 0.1 M Tris-HCl, pH 8.0, containing 0.9 M NaCI, 1 mM 2-mercaptoethanol and 0.5 mM EDTA. The suspension was shaken at 30°C for 20 min, then centrifuged for 10 min at 6500 X g (Step 1). The supernatant fluid containing the released proteins was concentrated to 19 ml using XM 50 Amicon ultrafilter (Step 2). The concentrated shock fluid was adjusted to pH 7.0 with 1 M potassium phosphate buffer, pH 7.0, and applied to DEAE-cellulose column (2.3 × 12 cm) which was previously equilibrated with 0.05 M potassium phosphate buffer, pH 7.0, and washed with 115 ml of the same buffer. All of the thiamine-binding activity from the shock fluid was absorbed to the column. Elution was carried out with a linear gradient consisting of 140 ml of the buffer in the mixing flask and an equal volume of buffer containing 0.2 M KCI in the reservoir. Fig. 1 shows a typical DEAE-cellulose chromatography elution profile obtained using shock fluid protein from S. cerevisiae. The thiamine-binding protein peak was reproducibly eluted at approximately 0.12 M KCI (Step 3). The DEAE-cellulose fractions were combined, and then concentrated and washed with the buffer on a XM 100A Amicon ultrafilter several times (Step 4). Table I shows the summary of the purification of a thiamine-binding protein from S. cerevisiae. The specific activity of the purified protein represents an overall purification of 7.7 fold with a recovery of 42.2 per cent. Disc gel electrophoresis at pH 8.9 of a purified thiamine-binding protein revealed a single band, and the binding activity toward thiamine was coincident with the stained part of the gel in the duplicate runs of the purified protein (data not shown). The molecular weight of the thiamine-binding protein was estimated to be 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis
~IIlOOIO'lO
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g i o o.o5
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25
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30 40 50 Fractionnumber
60
70
80
F i g . 1 . O E A E - c e i l u l o s e c o l u m n c h r o m a t o g r a p h y o f t h i a m i n e - b i n d i n g p r o t e i n f r o m Saccharomycee cerevisiae. 1 9 m l o f S t e p 2 w e r e a p p l i e d t o t h e c o l u m n ( 2 . 3 X 1 2 c m ) , w h i c h w a s e q u i l i b r a t e d w i t h 0.05 M potassium phosphate buffer, pH 7.0. After the washing with 115 ml of the buffer thiamineb i n d i n g p r o t e i n w a s e l u t e d w i t h a c o n c e n t r a t i o n g r a d i e n t starting f r o m 0 . 0 5 M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7 . 0 ( 1 4 0 m l ) t o t h e final b u f f e r w h i c h c o n t a i n e d 0 . 2 M KCI in t h e s a m e b u f f e r ( 1 4 0 ml). T h e f l o w r a t e w a s 3 0 m l p e r h o u r and f r a c t i o n v o l u m e w a s 5 ml. AH t h e p r o c e d u r e w a s carried o u t a t 4 ° C . T h e a c t i v i t y o f t h i a m i n e o b i n d / n g p r o t e i n w a s assessed b y a n e q u i l i b r i u m d i a l y s i s e x p e r i m e n t as p r e v i o u s l y d e s c r i b e d [ 3 ] . P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y the F o l i n m e t h o d [ 7 ] .
219 using the standard protein kit obtained from Boehringer Mannheim Company, and the protein was also stainable with periodic acid-Schiff suggesting that the yeast thiamine-binding protein is a glycoprotein. Finally, the electrophoretic pattern of a concentrated shock fluid from the yeast cells grown in the absence or presence of 1 pM thiamine was investigated. As shown in Fig. 2B at least eight protein bands were observed on standard disc gel electrophoresis in which a slowest moving broad band was coincident to thiamine-binding protein. However, the thiamine-binding protein was hardly detectable in the shock fluid from the cells grown in the presence of 1/~M thiamine (Fig. 2A).
A
B
F i g . 2 . Disc gel e l e c t x o p h o r e s i s o f c o n c e n t r a t e d s h o c k fluid f r o m S a c c h a r o m y c e s cercvislae g r o w n in t h e p r e s e n c e or a b s e n c e o f t h i a m i n e . 9 5 ~ g e a c h o f c o n c e n t r a t e d s h o c k f l u i d f r o m S. cerevislae g r o w n in t h e p r e s e n c e ( A ) o r a b s e n c e o f 1 / ~ M t h i a m i n e (B) w e r e r u n at 6 . 0 m A p e r t u b e f o r 1 h a t r o o m t e m p e r a t t t r e . T h e p r o t e i n w a s stained w i t h C o o m a s s i e Brilliant blue. TABLE I PURIFICATION
OF THIAMINE-BINDING
PROTEIN FROM SACCHAROMYCES
Step
Volume (ml)
Total binding activity (pmol thiamine bound)
Total protein (mg)
1. S h o c k f l u i d 2. C o n c e n t r a t e d s h o c k fluid 3. D E A E - c e l i n l o s e 4. U l t r a f i l t r a t i o n
400 19
8302.4 7125.8
6.2 2.7
50 50
3609.4 3506.0
0.43 0.34
Specific binding activity (Pmol thiamine bound]rag)
CEREVISIAE Recovery (%)
1 339.1 2 639.2
100 85.8
8 394.0 10 311.7
43.5 42.2
220
These results clearly show that the formation of a thiamine-binding protein in growing yeast cells could be repressed by exogenous thiamine in the growth medium as previously suggested [3]. Although a thiamine-binding protein released by cold osmotic shock from S. cerevisiae might be involved in thiamine transport in S. cerevisiae, further examination of the physical and chemical properties of this protein should provide necessary tools to examine in molecular terms, its role on thiamine transport in S. cerevisiae. We wish to express our thanks to Professor Yoshitsugu Nose for his interest in the present investigation. This woxk was supported in part by grants from the Ministry of Education of Japan.
References 1 2 3 4 5 6 7
Iwashima, A., N/sh/no, H. and Nose, Y. (1973) Biochlm. Biophys. Acta 330, 222--234 Iwash/ma, A., Wakabayashi, Y. and Nose, Y, (1975) Bioehim. Biophys. Acta 413, 243--247 lwashlma, A. and Nose, Y. (1976) J. Bacteriol. 128, 855--857 H o t , k , J. and Kotyk, A. (1973) Eur. J. Biochem. 32, 36--41 Schwencke, J., Farias, G. and Rojas, M. (1971) Eur. J. Biochem. 21,137--143 Wiekerham, L.J. (1951) U.S. Dept. Agric. Tech. Bull. 1029, 1--56 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275
BBA Report
BBA 31270 ISOLATION OF A THIAMINE-BINDING P R O T E I N F R O M SACCHAROMYCES CEREVISIAE
AKI0 IWASHIMA AND HIROSHI NISHIMURA Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamikyolzu, Kyoto (Japan) (Received November 6th, 1978) Key words: Thiamine-binding protein; (Saccharomyces cerevisiae)
Summary Thiamine-binding protein was isolated from Saccharomyces cerevisiae by successive procedures of cold osmotic shock treatment, DEAE-cellulose chromatography and ultrafiltration. The purified thiamine.binding protein was an electrophoretically homogeneous molecule which appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. No thiamine-binding protein was observed by disc gel electrophoresis in the shock fluid released from yeast ceils grown in the presence of I gM thiamine, indicating that the formation of this protein is regulated by exogenous thiamine as previously suggested.
Previous studies have shown that the transport of thiamine in Saccharomyces cerevisiae occurs by a carrier-mediated active process which is specific for thiamine [1,2]. It was also found that there exists a thiamine-binding protein in sonic extract of S. cerevisiae and it appeared to be repressed by exogenous thiamine with a concomitant decrease in thiamine uptake by yeast cells [3]. Purification of binding proteins from yeast cells,however, has been hampered by technical difficulties[4,5]. This paper describes the isolation of a thiamine-binding protein from S. cerevisiae. S. cerevisiae obtained as previously reported [2] was grown in 12 liters of thiamine-deficient Wickerham's minimal medium [6] at 30 ° C. They were harvested at the late exponential phase, and the cells were washed
218
twice with distilled water, then subjected to the cold osmotic shock treatment as follows. Washed yeast cells were suspended in 400 ml of 0.1 M Tris-HCl, pH 8.0, containing 0.9 M NaCI, 1 mM 2-mercaptoethanol and 0.5 mM EDTA. The suspension was shaken at 30°C for 20 min, then centrifuged for 10 min at 6500 X g (Step 1). The supernatant fluid containing the released proteins was concentrated to 19 ml using XM 50 Amicon ultrafilter (Step 2). The concentrated shock fluid was adjusted to pH 7.0 with 1 M potassium phosphate buffer, pH 7.0, and applied to DEAE-cellulose column (2.3 × 12 cm) which was previously equilibrated with 0.05 M potassium phosphate buffer, pH 7.0, and washed with 115 ml of the same buffer. All of the thiamine-binding activity from the shock fluid was absorbed to the column. Elution was carried out with a linear gradient consisting of 140 ml of the buffer in the mixing flask and an equal volume of buffer containing 0.2 M KCI in the reservoir. Fig. 1 shows a typical DEAE-cellulose chromatography elution profile obtained using shock fluid protein from S. cerevisiae. The thiamine-binding protein peak was reproducibly eluted at approximately 0.12 M KCI (Step 3). The DEAE-cellulose fractions were combined, and then concentrated and washed with the buffer on a XM 100A Amicon ultrafilter several times (Step 4). Table I shows the summary of the purification of a thiamine-binding protein from S. cerevisiae. The specific activity of the purified protein represents an overall purification of 7.7 fold with a recovery of 42.2 per cent. Disc gel electrophoresis at pH 8.9 of a purified thiamine-binding protein revealed a single band, and the binding activity toward thiamine was coincident with the stained part of the gel in the duplicate runs of the purified protein (data not shown). The molecular weight of the thiamine-binding protein was estimated to be 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis
~IIlOOIO'lO
.'/J'''
, 0.2 ~
g i o o.o5
-o., o
.! ~
25
O
~
I0
20
30 40 50 Fractionnumber
60
70
80
F i g . 1 . O E A E - c e i l u l o s e c o l u m n c h r o m a t o g r a p h y o f t h i a m i n e - b i n d i n g p r o t e i n f r o m Saccharomycee cerevisiae. 1 9 m l o f S t e p 2 w e r e a p p l i e d t o t h e c o l u m n ( 2 . 3 X 1 2 c m ) , w h i c h w a s e q u i l i b r a t e d w i t h 0.05 M potassium phosphate buffer, pH 7.0. After the washing with 115 ml of the buffer thiamineb i n d i n g p r o t e i n w a s e l u t e d w i t h a c o n c e n t r a t i o n g r a d i e n t starting f r o m 0 . 0 5 M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7 . 0 ( 1 4 0 m l ) t o t h e final b u f f e r w h i c h c o n t a i n e d 0 . 2 M KCI in t h e s a m e b u f f e r ( 1 4 0 ml). T h e f l o w r a t e w a s 3 0 m l p e r h o u r and f r a c t i o n v o l u m e w a s 5 ml. AH t h e p r o c e d u r e w a s carried o u t a t 4 ° C . T h e a c t i v i t y o f t h i a m i n e o b i n d / n g p r o t e i n w a s assessed b y a n e q u i l i b r i u m d i a l y s i s e x p e r i m e n t as p r e v i o u s l y d e s c r i b e d [ 3 ] . P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y the F o l i n m e t h o d [ 7 ] .
219 using the standard protein kit obtained from Boehringer Mannheim Company, and the protein was also stainable with periodic acid-Schiff suggesting that the yeast thiamine-binding protein is a glycoprotein. Finally, the electrophoretic pattern of a concentrated shock fluid from the yeast cells grown in the absence or presence of 1 pM thiamine was investigated. As shown in Fig. 2B at least eight protein bands were observed on standard disc gel electrophoresis in which a slowest moving broad band was coincident to thiamine-binding protein. However, the thiamine-binding protein was hardly detectable in the shock fluid from the cells grown in the presence of 1/~M thiamine (Fig. 2A).
A
B
F i g . 2 . Disc gel e l e c t x o p h o r e s i s o f c o n c e n t r a t e d s h o c k fluid f r o m S a c c h a r o m y c e s cercvislae g r o w n in t h e p r e s e n c e or a b s e n c e o f t h i a m i n e . 9 5 ~ g e a c h o f c o n c e n t r a t e d s h o c k f l u i d f r o m S. cerevislae g r o w n in t h e p r e s e n c e ( A ) o r a b s e n c e o f 1 / ~ M t h i a m i n e (B) w e r e r u n at 6 . 0 m A p e r t u b e f o r 1 h a t r o o m t e m p e r a t t t r e . T h e p r o t e i n w a s stained w i t h C o o m a s s i e Brilliant blue. TABLE I PURIFICATION
OF THIAMINE-BINDING
PROTEIN FROM SACCHAROMYCES
Step
Volume (ml)
Total binding activity (pmol thiamine bound)
Total protein (mg)
1. S h o c k f l u i d 2. C o n c e n t r a t e d s h o c k fluid 3. D E A E - c e l i n l o s e 4. U l t r a f i l t r a t i o n
400 19
8302.4 7125.8
6.2 2.7
50 50
3609.4 3506.0
0.43 0.34
Specific binding activity (Pmol thiamine bound]rag)
CEREVISIAE Recovery (%)
1 339.1 2 639.2
100 85.8
8 394.0 10 311.7
43.5 42.2
220
These results clearly show that the formation of a thiamine-binding protein in growing yeast cells could be repressed by exogenous thiamine in the growth medium as previously suggested [3]. Although a thiamine-binding protein released by cold osmotic shock from S. cerevisiae might be involved in thiamine transport in S. cerevisiae, further examination of the physical and chemical properties of this protein should provide necessary tools to examine in molecular terms, its role on thiamine transport in S. cerevisiae. We wish to express our thanks to Professor Yoshitsugu Nose for his interest in the present investigation. This woxk was supported in part by grants from the Ministry of Education of Japan.
References 1 2 3 4 5 6 7
Iwashima, A., N/sh/no, H. and Nose, Y. (1973) Biochlm. Biophys. Acta 330, 222--234 Iwash/ma, A., Wakabayashi, Y. and Nose, Y, (1975) Bioehim. Biophys. Acta 413, 243--247 lwashlma, A. and Nose, Y. (1976) J. Bacteriol. 128, 855--857 H o t , k , J. and Kotyk, A. (1973) Eur. J. Biochem. 32, 36--41 Schwencke, J., Farias, G. and Rojas, M. (1971) Eur. J. Biochem. 21,137--143 Wiekerham, L.J. (1951) U.S. Dept. Agric. Tech. Bull. 1029, 1--56 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275
