YEAST
VOL.
7: 849-855 (1991)
The Cysteine Transport System of Saccharornyces cerevisiae BUh-ICHIRO ONO* A N D KAZUHIDE NAlTO Lohorutorj. of En vironnienrul Hj*gicwe Chemistry. I;clcultj~of Pharmaceutical Sciences. O k a y m u Universit.v, Oh ( I I aniu 700, Japan
Received 9 April I99 1 ; reviscd 3 June 1991
Although Socchuromyces ccwvisiue strains had different cystcine uptake activities, thcy revealed monophasic uptake kinetics and had thc samc KT (83.3 p ~ ) .The optimal ptI ofcysteinc uptake was between 4.5 and 5.0.but thc activity was quickly lost if cells wcrc kept in buffer. Whcn the activity was measured in the growth medium, it increased in the prcsencc of EDTA and greatly decreased in the presencc of mercuricchloride. Thioglycol as well as metabolic inhibitors such as dinitrophcrol and azidc wcrc inhibitory. Homocystcine and mcthionine werc competitivc and non-competitive inhibitors. respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, weconclude that the system mediating uptake of cystcine is specific (we thus name it thc cysteine transport system) and that the cystcine transport system recognizes not only the SH-group but also amino- and carboxyl-groups. In w,ild-typc strains the cysteine transport system was derepressed only when the cells wcrc incubated without any sulfur source. On thc other hand, in cysteine-dependent mutants. cystcine uptake activity increased with increase of cxogenous supply of cystcine, glutathione or methioninc. From this rcsult. we suspect that the cellular cysteine lcvel is the limiting factor for biosynthesis of the cysteine transport system in cysteine-dependent strains. K F Y WORDS - Sacchoromycc..s cerevisiae; amino
acid transport; Cysteine.
INTRODUCTION
MATERIALS A N D METHODS
Although cysteine plays unique physiological roles due to its possession of an SH-group (mediation of oxdation:'reduction reactions, radical scavenge, etc). regulation of its cellular level is not fully understood. Recently, it has been shown that Succhurom!ws cerevisiue has two functional cysteine biosynthetic pathways: one utilizes sulfatesulfur while the other utilizes homocysteine-sulfur (Ono er ul.. 1984. 1988). In the latter pathway, cystathionine y-lyase but not cystathionine psynthase is repressed by cysteine o r glutathione (On0 cJr al., 1991). In the course of the study, we have found that the cysteine uptake activity, which differs from one strain to another, affects regulation of the enzyme. I t is natural to assume that the cellular cysteine level is determined not only by biosynthesis but also by uptake. We therefore decided to characterize the 5. cerevisiae cysteine transport system. Here, we present the results of our study and the conclusion that the cysteine transport system is regulated by cellular cysteine.
Strains Strains used in this study are listed in Table 1. S288C is a widely used prototroph (Mortimer, 1987), IS66-4C is a prototroph used in this laboratory (On0 er ul., 1988), and WT is a prototroph from which various methionine-dependent mutations including metf 7 were isolated (Naiki and Iwata, 1962). IS66-3B is a strain closely related to IS66-4C, and was found to havea cys2-2mutation (On0 et ul., 1988). NA12-3C (cys2-f cys4-I) and NA22-10C (metf7) are genetically very similar to IS66-4C (On0 et ul., 1988. 1991). NA12-3C (cys2 cys4) grew only when cysteine was supplied, while Na22-10C (metf7) grew when either cysteine o r methionine was supplied. These strains grew better on supplementation of glutathione (y-glutamyl-cysteinylglycine) than cysteine. Thus, we used glutathione as the organic sulfur source to grow cells unless indicated ot herwise.
*Addressee for correspondence. 0749 503X.91;(I80849 07 $05.00 0 1991 by John Wiley &Sons Ltd
Grontth conditions YPD medium (2% glucose, 2% peptone and 1 % yeast extract) was used for routine growth of cells
850
B. O N 0 AND K. NAITO
Table I . Strains used in this study Strain S288C WT IS66-4C IS66-3B NA12-3C NA22-10C
Genotype
Reference
MATa M A Ta MATa MATa cys2-2 M A T a cys2-1 cysl-1 MATa met17
(Sherman et al., 1986). For cysteine transport experiments, a synthetic (SD) medium was used (Wickerham, 1956). Sulfur-free medium was made by substituting sulfate salts of SD medium with chloride salts (On0 et al., 1991). Growth temperature was 30°C. Cell density
A portion of culture was added to an equal volume of 10% trichloro-acetic acid. The mixture was kept in an ice bath for at least 1 hr. The optical density at 600 nm was then measured.
Mortimer (1987) Naiki and Iwata (1 962) Ono et al. (1 988) Ono et al. (1 988) Ono et al. (1988) Ono et al. (1988)
Japan). Radioactivity was measured with a liquid scintillation counter (Aloka model LSC-901; Aloka, Mitaka, Tokyo, Japan). In order to obtain statistically meaningful data, more than 1000 counts were measured for each sample. Radioactivity taken up by cells increased linearly for 7 min or longer, and the initial rate was estimated from the slope of the linear portion of the plot. Specific activity of trans ort was represented by nmol (OD60,cell unit)- min - I .
P
'
Kinetic analyses of cysteine transport activity
To measure KT and J,,,,,, cells were grown as described above. To 5 ml of the cell culture, various Cells were grown for 14 hr in YPD medium. The volumes of 5 m ~ - [ ' ~ C ( U) ] ~ - c y ste in (2.96 e GBq cells were harvested, washed twice with water, mol-'), were added. The volume of the reaction suspended in SD medium containing 130 p ~ - L - mixture was adjusted to 5.5 ml with water. Radioglutathione, and incubated for 10hr. The cells activity in the cells was then measured as described were sedimented with a table-top centrifuge, re- above. To study the mode of inhibition, a fixed suspended in the fresh growth medium, and in concentration (100 p ~ )of an inhibitor, D,Lincubated for 4 hr. By this procedure, cells in the homocysteine or L-methionine, was added to the logarithmic phase were obtained. The final cell reaction mixture containing various concentrations density of the culture was about 1.5 OD,,,; one unit of ["C(U)] L-cysteine (2.96 GBq mol"). Radioof OD,,, corresponds to 7.0 x lo7 cells ml-I. The activity of the cells was then measured. cysteine transport activity was measured at 30°C in the culture medium. The culture medium was used without pH adjustment; the pH of the growth Chemicals medium was 5.0 and 2.6 before and after growth of Peptone and yeast extract were products of Difco cells, respectively. The reaction was started by adding 0.5ml of Laboratories (Detroit, Michigan, USA). [14C(U)] [I4C(U)] L-cysteine (0.5 mM; 2.96 GBq mol-I) and L-cysteine was purchased from Amersham 5 ml of cell culture. After intervals, 1 mi samples International plc (Amersham, Buckinghamshire, were withdrawn. Each sample was filtrated through England). Amino acids were products of Wako a nitrocellulose membrane (pore size 0.45 pm; Toyo Pure Chemicals Co. Ltd (Osaka, Japan) or Nakarai Roshi Ltd, Tokyo, Japan). The cells trapped on the Chemicals Co. Ltd (Kyoto, Japan). Cysteic acid membrane were washed twice with 4 ml of 10 mM-L- was kindly supplied by Dr S. Ohmori (Faculty of cysteine. The membrane harboring the cells was Pharmaceutical Sciences, Okayama University, dried and immersed in 5ml of scintillation liquid Okayama, Japan). Other chemicals used were (EX-H; Wako Pure Chemicals Co. Ltd, Osaka, analytical grades. Cysteine transport activity
+
85 1
CYSTEINE TRANSPORT
RESULTS C),.stc+c.
uptake activity of various strains
Strains were grown to logarithmic phase in SD medium containing 130 p4-glutathione, and cysteine taken up by the cells was measured (Table 2). All prototrophs tested, S288C. WT and IS66-4C. did not take up cysteine well. IS66-3B (cys-2-2) was also low in cysteine uptake activity. In contrast, strains NA22-10c(metl7)and NA12-3C(cy.s2cys4) had substantial activities of cysteine uptake. Thus, wc cxamincd these strains further; IS66-4C (wild type) was used as a control since it is a prototroph closely related genetically to them (Ono et al., 1988, 1991). Table 2.
Cystcinc uptake activities of various strains
Strain
Initial velocity (nmol m i n - ' OD,,
I
/
I
/
I
I
I
Figure I . Kinetic analyses of cysteine uptake of three strains. Strains NA22-10C (mrtl7)(C). NA12-3C (cys2 c y . 4 ( L )and IS66-4C (wild type) (LI) were grown in SD medium containing 130 pM-glutathione. and cysteine uptake activity was assayed at various cysteine concentrations (see Materials and Methods).
I)
Table 3. Kinetic values of the cystcine transport system
~
S288C (wild type) WT (wild type) IS66-4C (wild type) IS66-3B ( ~ 1 . ~ 2 ) Na 12-3C: ( ~ ' 1 .~~' 21 . ~ 4 ) NA22-IOC (rncv17) NA22- I OC (hcat treated)*
X
0.15
0.10 0.17 0.12 0.30 0.78
0.00
'Cells were incubated in a boiling water-bath for 5 min prior to the assay.
K.1 Strain
(PM)
NA22-10C (met17 N A 12-3C (c.v.s~C Y S ~ ) IS66-4C (wild type)
83.3 83.3 83.3
Jm,,
(nmol m i n - '
OD;
I)
3.33 0.9 1 1.25
Optimal pH of the cysteine transport system Kinetics of'qvsteine transport Cysteine uptake activity was completely lost if cells were heated in a boiling water bath for 5 min (Table 2). The result indicates that the activity we measured was transport of cystcine into the cell rather than mere binding of cysteine to the cell wall. Next, we investigated the kinetic characteristics of the cystcine transport system (Figure I). The Lineweaver-Burk plots of cysteine transport of the three strains, NA22-10C (mrll7). Na12-3C (cys2 cys4) and IS66-4C (wild type), were linear and monophasic. It is thusconcluded that cysteine transport is mediated by a single system or by systems of similar affinity to cysteine in each strain. The three strains differed from one another in terms of J,,,,,, but they had thc same K, (Table 3). Strain NA22-10C ( m ~ t 1 7had ) the highest J,,,, and we used this strain in the subsequent experiments.
Strain NA22-10C (metl7) was grown in SD medium supplemented with I30 pM-glutathione. Cysteine transport activity was then measured at various pH values (Figure 2). The cells retained the cysteine transport shortly after the transfer to the buffer, but the activity was 56% or less of that in the culture medium; the highest activity was obscrved at pH 4.5-5.0. The cells lost activity if they were incubated in the buffer for 4 hr. Because we were interested in cysteine transport of the growing cells, we subsequently achieved cysteine uptake experiments in the culture medium. Eflects oj'various chemicals on the cysteine transport system We examined the effects of various chemicals on cysteine transport activity using NA22- IOC (metl7) (Table 4). The activity increased about 1.5-fold in
852
B. ON0 AND K. NAlTO
.-.I
0-Q
2
6
4
8
growth medium increases. The result suggests that biosynthesis of the cysteine transport system is affected by the cellular cysteine level; increasing the supply of L-cysteine, directly or via L-methionine or L-glutathione, increases biosynthesis of the cysteine transport system. The explanation of this result is not straightforward because we found that the cysteine transport system was repressed by cysteine (see below).
PH Figure 2. pH dependence of cysteine uptake activity of strain NA22-10C (rner17). Cells were grown in SD medium containing 130 pwglutathione to mid-logarithmic phase, harvested, washed twice with water, and then suspended in 20 mwcitrate buffer (pH 2.5-5.5) or 20 m ~ - p h o s p h a t e buffer (pH 6.G8.0); cysteine uptake activity was measured soon after (0)and 4 h after ( 0 ) the transfer.
the presence of EDTA, suggesting that the cysteine transport system is inhibited by metal ions present in the growth medium. The activity was markedly reduced by a strong acid (H,SO,) and an oxidizing agent (KMnO,). HgCI, was also a potent inhibitor. Thioglycol (2-mercaptoethanol) was more inhibitory than ethanol, indicating the importance of the SH-group for the function of the cysteine transport system. However, ammonium sulfate was not inhibitory. Homocysteine was inhibitory, but cysteamine and cysteic acid were not effective inhibitors at the concentrations we tested. Cystine, glutathione and cystathionine were not inhibitory either. From these results, we conclude that the transport system for cysteine is specific and it recognizes the SH-group as well as amino- and carboxylgroups. Hereafter, we call this transport system, the cysteine transport system. The cysteine transport system is inhibited by methionine and metabolic inhibitors such as dinitrophenol, NaN, and KCN. It should be added that inhibition due to homocysteine and methionine is competitive and non-competitive, respectively (Figure 3). Effects of suljiur compounds on the hiosynthesis of the cysteine transport system NA22-10C (metl7) was grown in SD medium supplemented with various concentrations of organic sulfur compound, L-cysteine, L-methionine or L-glutathione, and then cysteine transport activity was measured (Figure 4). The result clearly shows that cysteine transport activity of the cells increases as the supply of these compounds to the
Derepression of the cysteine transport system of strain IS66-4C It was puzzling that strain IS66-4C (wild type) had very low cysteine uptake activity (see Table 2). We thought that the result would be due to repression of the cysteine transport system. We grew strain IS66-4C overnight in liquid YPD medium and then transferred it to sulfur-free liquid medium. After intervals, we assayed cysteine uptake activity (Figure 5). The activity quickly increased after the shift, reached the highest level after 30 min and gradually decreased thereafter. The increase of activity is attributed to derepression of the cysteine transport system. In fact the increase of activity was not observed if cycloheximide was added to YPD medium 15 min prior to the transfer to SD medium. The subsequent decrease of cysteine uptake activity is attributed to the absence of metabolism, which, as mentioned previously, leads to the loss of cysteine uptake activity. DISCUSSION S . cerevisiae takes up amino acids via transport systems of various specificity. One of them, known as ‘general amino acid permease’ (GAP; Grenson et al., 1970), mediates transport of many amino acids. Although there has been no direct evidence that the GAP system mediates cysteine transport, data presented by Greasham and Moat (1973) strongly suggest that it does so. However, it is known that this system is inhibited by ammonium ions (Grenson et al., 1970). The cysteine uptake observed in the present experimental conditions is therefore not attributable to the GAP system. Moreover, cysteine uptake is competitively inhibited by homocysteine (Figure 3), indicating that the transport system we are dealing with is specific to sulfhydryl amino acids; we refer to it as the cysteine transport system. It should be stressed here that the cysteine transport system rapidly loses its
853
CYSTEINE TRANSPORT
Table 4. Effects of various chemicals on cysteine uptake* Concentration Chemical None EDTA HW4 Acetic acid KMnO, HgCh Thioglycol Ethanol (NH,),SO, L-Cystine D,L-Homocysteine Cysteic acid Cysteamine L-Glutathione L-Cystathionine L-Methionine DNP NaN, KCN
(PM)
100
1000 3130 3130 100 37 2600 3130 100 100 100 1000 1000 100 100 100 100 1000 100 1000 1000
Activity (nmol min-' OD6oo-')
Relative activity (YO) 100
0.78 f0.05 1.22k0.01 1.22 fO.01 0.38 f0.10 0.82 f0.09 0.45 f0.04 0.08 f0.03 0.43 f0.11 0.52 f0.00 0.93 f0.1 1 0.97 f0.12 0.67 f0.04 0.80 f0.12 0.90 f0.18 0.83 f0.07 0.78 f0.05 0.48 f0.03 0.2 1 k0.05 0.02 f0.00 0.13 f0.02 0.03 f0.00 0.42 0.08
155
155 49 104 57 10 55 66 119 123 85 102 115
106 100 62 28 2 17 4
53
*Strain NA22-10C (metl7) was used. Cysteine uptake was assayed in the presence of 100 PMcysteine and the indicated chemicals at the indicated concentration. DNP, Dinitrophenol.
.-C
E
x
4-
3-
T.
0
E
v
?o
2-
.= .-> c
v
0
o
x
.-v '5
0.8
854
B. O N 0 AND K. NAITO
a , t
2.5
%
0.0
I
0
1
I
7 4 - b
2 3 Time ( h r )
15
48
Figure 5. Derepression of the cysteine transport system. Strain IS66-4C (wild type) was grown in YPD liquid medium, harvested, washed twice with water, suspended in sulfur-free liquid medium, and incubated at 30°C with rotary shaking (60 rpm). After intervals, cysteine transport activity was assayed. The cells were treated with (0)or without ( 0 )50 pg/ml cycloheximide; cycloheximide was added to Y P D medium 15 min before the transfer and to sulfur-free medium to which the cells were transferred.
activity in buffer (Figure 1). From this observation, together with the effective inactivation of the system by metabolic inhibitors (Table 4), we conclude that the system depends on energy metabolism. We have found that the cysteine transport system is non-competitively inhibited by methionine (Figure 3). According to Gits and Grenson (1967), methionine transport is not inhibited by cysteine. Therefore, it appears as if S. cerevisiue takes up methionine preferentially over cysteine. As we have argued in the preceding paper (On0 et ul., 1991), S . cerevisiue appears to be programmed to utilize methionine preferentially if it is available from the environment. The present finding indicates that the regulation of transport systems operates under the same principle. It has been found that cysteine transport activity is low in prototrophic strains, which may be the reason why attention has not previously been drawn to the cysteine transport system. We have found that cysteine-dependent mutants are higher in cysteine uptake activity than the prototrophic strains. We have clearly shown that the cysteine transport system is repressed in the prototrophic strains and that derepression of the system takes place if the strains are incubated in medium lacking any sulfur source. The result that cysteine transport activity of the cysteine-dependent strains increased with increasing supply of organic sulfur sources is contradictory to our present argument. Our rationale at the moment is as follows. The cellular cysteine level required for repression of the cysteine uptake
system is much higher than that for cystathionine y-lyase. So that, under conditions where cystathionine y-lyase is repressed, the cysteine transport system remains derepressed. In such circumstances, increasing the supply of cysteine results in the increased biosynthesis of the cysteine transport system if cysteine is the limiting factor. That is, our speculation predicts that the cysteine transport system is rich in cysteine. Inhibition of the cysteine transport system by thioglycol (Table 4) favors the presence of SH-group(s) in the cysteine transport system. Strong inhibition by HgCl, (Table 4) may also be taken as another indication that the cysteine transport system contains the SH-group. However, there is a possibility that HgCl, inhibits some other biological functions such as energy metabolism, which then affects the cysteine transport activity. Anyway, it is important to know the amino acid composition of the cysteine transport system to support our hypothesis. ACKNOWLEDGEMENTS We thank D r S. Ohmori (Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan) for supply of cysteic acid. We thank Dr S. Shinoda (Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan) for his valuable suggestions during the work. We also thank the staff of the Tsushima Radioisotope Laboratory of Okayama University, where the radioisotope experiments were carried out. REFERENCES Gits, J. J. and Grenson, M. (1967). Multiplicity of the amino acid permeases in Saccharomyces cercvisiae. 111. Evidence for a specific methionine-transporting system. Biochim Biophys. Acta 135,507-5 16. Greasham, R. L. and Moat, A. G. (1973). Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae. J . Bacteriol. 115,975-98 1. Grenson, M., Hous, C . and Crabeel, M. (1970). Multiplicity of the amino acid permease in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J . Bacteriol. 103,770-777. Mortimer, R. (1987). Yeast Genetic Stock Center CUUlogue. Yeast Genetic Center, University of California, Berkeley, California. Naiki, N. and Iwata, M. (1962). Utilization of sulfur compounds by methionineless mutants of Saccharomycrs cerevisiae. Sci.Rep. Fuc. Lib. Arts Educ., Glfu Univ. 3, 70-75. (In Japanese with an English summary.)
CYSTEINE TRANSPORT
Ono. B., Suruga, T., Yamamoto, M., Yamamoto, S., Murata, K., Kimura, A,, Shinoda, S. and Ohmori, S. (1984). Cystathionine accumulation in Saccharomyces cerevisiae. J . Bacteriol. 158,86&865. Ono, B., Shirahige, Y., Nanjoh, A,, Andou, N., Ohue, H. and Ishino-Arao, Y. (1988). Cysteine biosynthesis in Saccharomyces cerevisiae: mutation that confers cystathionine (3-synthase deficiency. J . Bacteriol. 170, 5883-5889.
855 Ono, B., Naito, K., Shirahige, Y. and Yamamoto, M. (1991). Regulation ofcystathionine y-lyasein Saccharomyces cerevisiae. Yeast 7,843-848. Sherman, F., Fink, G. R. and Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Wickerham, L. J. (1956). A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeast. J. Bacteriol. 52,293-301.
VOL.
7: 849-855 (1991)
The Cysteine Transport System of Saccharornyces cerevisiae BUh-ICHIRO ONO* A N D KAZUHIDE NAlTO Lohorutorj. of En vironnienrul Hj*gicwe Chemistry. I;clcultj~of Pharmaceutical Sciences. O k a y m u Universit.v, Oh ( I I aniu 700, Japan
Received 9 April I99 1 ; reviscd 3 June 1991
Although Socchuromyces ccwvisiue strains had different cystcine uptake activities, thcy revealed monophasic uptake kinetics and had thc samc KT (83.3 p ~ ) .The optimal ptI ofcysteinc uptake was between 4.5 and 5.0.but thc activity was quickly lost if cells wcrc kept in buffer. Whcn the activity was measured in the growth medium, it increased in the prcsencc of EDTA and greatly decreased in the presencc of mercuricchloride. Thioglycol as well as metabolic inhibitors such as dinitrophcrol and azidc wcrc inhibitory. Homocystcine and mcthionine werc competitivc and non-competitive inhibitors. respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, weconclude that the system mediating uptake of cystcine is specific (we thus name it thc cysteine transport system) and that the cystcine transport system recognizes not only the SH-group but also amino- and carboxyl-groups. In w,ild-typc strains the cysteine transport system was derepressed only when the cells wcrc incubated without any sulfur source. On thc other hand, in cysteine-dependent mutants. cystcine uptake activity increased with increase of cxogenous supply of cystcine, glutathione or methioninc. From this rcsult. we suspect that the cellular cysteine lcvel is the limiting factor for biosynthesis of the cysteine transport system in cysteine-dependent strains. K F Y WORDS - Sacchoromycc..s cerevisiae; amino
acid transport; Cysteine.
INTRODUCTION
MATERIALS A N D METHODS
Although cysteine plays unique physiological roles due to its possession of an SH-group (mediation of oxdation:'reduction reactions, radical scavenge, etc). regulation of its cellular level is not fully understood. Recently, it has been shown that Succhurom!ws cerevisiue has two functional cysteine biosynthetic pathways: one utilizes sulfatesulfur while the other utilizes homocysteine-sulfur (Ono er ul.. 1984. 1988). In the latter pathway, cystathionine y-lyase but not cystathionine psynthase is repressed by cysteine o r glutathione (On0 cJr al., 1991). In the course of the study, we have found that the cysteine uptake activity, which differs from one strain to another, affects regulation of the enzyme. I t is natural to assume that the cellular cysteine level is determined not only by biosynthesis but also by uptake. We therefore decided to characterize the 5. cerevisiae cysteine transport system. Here, we present the results of our study and the conclusion that the cysteine transport system is regulated by cellular cysteine.
Strains Strains used in this study are listed in Table 1. S288C is a widely used prototroph (Mortimer, 1987), IS66-4C is a prototroph used in this laboratory (On0 er ul., 1988), and WT is a prototroph from which various methionine-dependent mutations including metf 7 were isolated (Naiki and Iwata, 1962). IS66-3B is a strain closely related to IS66-4C, and was found to havea cys2-2mutation (On0 et ul., 1988). NA12-3C (cys2-f cys4-I) and NA22-10C (metf7) are genetically very similar to IS66-4C (On0 et ul., 1988. 1991). NA12-3C (cys2 cys4) grew only when cysteine was supplied, while Na22-10C (metf7) grew when either cysteine o r methionine was supplied. These strains grew better on supplementation of glutathione (y-glutamyl-cysteinylglycine) than cysteine. Thus, we used glutathione as the organic sulfur source to grow cells unless indicated ot herwise.
*Addressee for correspondence. 0749 503X.91;(I80849 07 $05.00 0 1991 by John Wiley &Sons Ltd
Grontth conditions YPD medium (2% glucose, 2% peptone and 1 % yeast extract) was used for routine growth of cells
850
B. O N 0 AND K. NAITO
Table I . Strains used in this study Strain S288C WT IS66-4C IS66-3B NA12-3C NA22-10C
Genotype
Reference
MATa M A Ta MATa MATa cys2-2 M A T a cys2-1 cysl-1 MATa met17
(Sherman et al., 1986). For cysteine transport experiments, a synthetic (SD) medium was used (Wickerham, 1956). Sulfur-free medium was made by substituting sulfate salts of SD medium with chloride salts (On0 et al., 1991). Growth temperature was 30°C. Cell density
A portion of culture was added to an equal volume of 10% trichloro-acetic acid. The mixture was kept in an ice bath for at least 1 hr. The optical density at 600 nm was then measured.
Mortimer (1987) Naiki and Iwata (1 962) Ono et al. (1 988) Ono et al. (1 988) Ono et al. (1988) Ono et al. (1988)
Japan). Radioactivity was measured with a liquid scintillation counter (Aloka model LSC-901; Aloka, Mitaka, Tokyo, Japan). In order to obtain statistically meaningful data, more than 1000 counts were measured for each sample. Radioactivity taken up by cells increased linearly for 7 min or longer, and the initial rate was estimated from the slope of the linear portion of the plot. Specific activity of trans ort was represented by nmol (OD60,cell unit)- min - I .
P
'
Kinetic analyses of cysteine transport activity
To measure KT and J,,,,,, cells were grown as described above. To 5 ml of the cell culture, various Cells were grown for 14 hr in YPD medium. The volumes of 5 m ~ - [ ' ~ C ( U) ] ~ - c y ste in (2.96 e GBq cells were harvested, washed twice with water, mol-'), were added. The volume of the reaction suspended in SD medium containing 130 p ~ - L - mixture was adjusted to 5.5 ml with water. Radioglutathione, and incubated for 10hr. The cells activity in the cells was then measured as described were sedimented with a table-top centrifuge, re- above. To study the mode of inhibition, a fixed suspended in the fresh growth medium, and in concentration (100 p ~ )of an inhibitor, D,Lincubated for 4 hr. By this procedure, cells in the homocysteine or L-methionine, was added to the logarithmic phase were obtained. The final cell reaction mixture containing various concentrations density of the culture was about 1.5 OD,,,; one unit of ["C(U)] L-cysteine (2.96 GBq mol"). Radioof OD,,, corresponds to 7.0 x lo7 cells ml-I. The activity of the cells was then measured. cysteine transport activity was measured at 30°C in the culture medium. The culture medium was used without pH adjustment; the pH of the growth Chemicals medium was 5.0 and 2.6 before and after growth of Peptone and yeast extract were products of Difco cells, respectively. The reaction was started by adding 0.5ml of Laboratories (Detroit, Michigan, USA). [14C(U)] [I4C(U)] L-cysteine (0.5 mM; 2.96 GBq mol-I) and L-cysteine was purchased from Amersham 5 ml of cell culture. After intervals, 1 mi samples International plc (Amersham, Buckinghamshire, were withdrawn. Each sample was filtrated through England). Amino acids were products of Wako a nitrocellulose membrane (pore size 0.45 pm; Toyo Pure Chemicals Co. Ltd (Osaka, Japan) or Nakarai Roshi Ltd, Tokyo, Japan). The cells trapped on the Chemicals Co. Ltd (Kyoto, Japan). Cysteic acid membrane were washed twice with 4 ml of 10 mM-L- was kindly supplied by Dr S. Ohmori (Faculty of cysteine. The membrane harboring the cells was Pharmaceutical Sciences, Okayama University, dried and immersed in 5ml of scintillation liquid Okayama, Japan). Other chemicals used were (EX-H; Wako Pure Chemicals Co. Ltd, Osaka, analytical grades. Cysteine transport activity
+
85 1
CYSTEINE TRANSPORT
RESULTS C),.stc+c.
uptake activity of various strains
Strains were grown to logarithmic phase in SD medium containing 130 p4-glutathione, and cysteine taken up by the cells was measured (Table 2). All prototrophs tested, S288C. WT and IS66-4C. did not take up cysteine well. IS66-3B (cys-2-2) was also low in cysteine uptake activity. In contrast, strains NA22-10c(metl7)and NA12-3C(cy.s2cys4) had substantial activities of cysteine uptake. Thus, wc cxamincd these strains further; IS66-4C (wild type) was used as a control since it is a prototroph closely related genetically to them (Ono et al., 1988, 1991). Table 2.
Cystcinc uptake activities of various strains
Strain
Initial velocity (nmol m i n - ' OD,,
I
/
I
/
I
I
I
Figure I . Kinetic analyses of cysteine uptake of three strains. Strains NA22-10C (mrtl7)(C). NA12-3C (cys2 c y . 4 ( L )and IS66-4C (wild type) (LI) were grown in SD medium containing 130 pM-glutathione. and cysteine uptake activity was assayed at various cysteine concentrations (see Materials and Methods).
I)
Table 3. Kinetic values of the cystcine transport system
~
S288C (wild type) WT (wild type) IS66-4C (wild type) IS66-3B ( ~ 1 . ~ 2 ) Na 12-3C: ( ~ ' 1 .~~' 21 . ~ 4 ) NA22-IOC (rncv17) NA22- I OC (hcat treated)*
X
0.15
0.10 0.17 0.12 0.30 0.78
0.00
'Cells were incubated in a boiling water-bath for 5 min prior to the assay.
K.1 Strain
(PM)
NA22-10C (met17 N A 12-3C (c.v.s~C Y S ~ ) IS66-4C (wild type)
83.3 83.3 83.3
Jm,,
(nmol m i n - '
OD;
I)
3.33 0.9 1 1.25
Optimal pH of the cysteine transport system Kinetics of'qvsteine transport Cysteine uptake activity was completely lost if cells were heated in a boiling water bath for 5 min (Table 2). The result indicates that the activity we measured was transport of cystcine into the cell rather than mere binding of cysteine to the cell wall. Next, we investigated the kinetic characteristics of the cystcine transport system (Figure I). The Lineweaver-Burk plots of cysteine transport of the three strains, NA22-10C (mrll7). Na12-3C (cys2 cys4) and IS66-4C (wild type), were linear and monophasic. It is thusconcluded that cysteine transport is mediated by a single system or by systems of similar affinity to cysteine in each strain. The three strains differed from one another in terms of J,,,,,, but they had thc same K, (Table 3). Strain NA22-10C ( m ~ t 1 7had ) the highest J,,,, and we used this strain in the subsequent experiments.
Strain NA22-10C (metl7) was grown in SD medium supplemented with I30 pM-glutathione. Cysteine transport activity was then measured at various pH values (Figure 2). The cells retained the cysteine transport shortly after the transfer to the buffer, but the activity was 56% or less of that in the culture medium; the highest activity was obscrved at pH 4.5-5.0. The cells lost activity if they were incubated in the buffer for 4 hr. Because we were interested in cysteine transport of the growing cells, we subsequently achieved cysteine uptake experiments in the culture medium. Eflects oj'various chemicals on the cysteine transport system We examined the effects of various chemicals on cysteine transport activity using NA22- IOC (metl7) (Table 4). The activity increased about 1.5-fold in
852
B. ON0 AND K. NAlTO
.-.I
0-Q
2
6
4
8
growth medium increases. The result suggests that biosynthesis of the cysteine transport system is affected by the cellular cysteine level; increasing the supply of L-cysteine, directly or via L-methionine or L-glutathione, increases biosynthesis of the cysteine transport system. The explanation of this result is not straightforward because we found that the cysteine transport system was repressed by cysteine (see below).
PH Figure 2. pH dependence of cysteine uptake activity of strain NA22-10C (rner17). Cells were grown in SD medium containing 130 pwglutathione to mid-logarithmic phase, harvested, washed twice with water, and then suspended in 20 mwcitrate buffer (pH 2.5-5.5) or 20 m ~ - p h o s p h a t e buffer (pH 6.G8.0); cysteine uptake activity was measured soon after (0)and 4 h after ( 0 ) the transfer.
the presence of EDTA, suggesting that the cysteine transport system is inhibited by metal ions present in the growth medium. The activity was markedly reduced by a strong acid (H,SO,) and an oxidizing agent (KMnO,). HgCI, was also a potent inhibitor. Thioglycol (2-mercaptoethanol) was more inhibitory than ethanol, indicating the importance of the SH-group for the function of the cysteine transport system. However, ammonium sulfate was not inhibitory. Homocysteine was inhibitory, but cysteamine and cysteic acid were not effective inhibitors at the concentrations we tested. Cystine, glutathione and cystathionine were not inhibitory either. From these results, we conclude that the transport system for cysteine is specific and it recognizes the SH-group as well as amino- and carboxylgroups. Hereafter, we call this transport system, the cysteine transport system. The cysteine transport system is inhibited by methionine and metabolic inhibitors such as dinitrophenol, NaN, and KCN. It should be added that inhibition due to homocysteine and methionine is competitive and non-competitive, respectively (Figure 3). Effects of suljiur compounds on the hiosynthesis of the cysteine transport system NA22-10C (metl7) was grown in SD medium supplemented with various concentrations of organic sulfur compound, L-cysteine, L-methionine or L-glutathione, and then cysteine transport activity was measured (Figure 4). The result clearly shows that cysteine transport activity of the cells increases as the supply of these compounds to the
Derepression of the cysteine transport system of strain IS66-4C It was puzzling that strain IS66-4C (wild type) had very low cysteine uptake activity (see Table 2). We thought that the result would be due to repression of the cysteine transport system. We grew strain IS66-4C overnight in liquid YPD medium and then transferred it to sulfur-free liquid medium. After intervals, we assayed cysteine uptake activity (Figure 5). The activity quickly increased after the shift, reached the highest level after 30 min and gradually decreased thereafter. The increase of activity is attributed to derepression of the cysteine transport system. In fact the increase of activity was not observed if cycloheximide was added to YPD medium 15 min prior to the transfer to SD medium. The subsequent decrease of cysteine uptake activity is attributed to the absence of metabolism, which, as mentioned previously, leads to the loss of cysteine uptake activity. DISCUSSION S . cerevisiae takes up amino acids via transport systems of various specificity. One of them, known as ‘general amino acid permease’ (GAP; Grenson et al., 1970), mediates transport of many amino acids. Although there has been no direct evidence that the GAP system mediates cysteine transport, data presented by Greasham and Moat (1973) strongly suggest that it does so. However, it is known that this system is inhibited by ammonium ions (Grenson et al., 1970). The cysteine uptake observed in the present experimental conditions is therefore not attributable to the GAP system. Moreover, cysteine uptake is competitively inhibited by homocysteine (Figure 3), indicating that the transport system we are dealing with is specific to sulfhydryl amino acids; we refer to it as the cysteine transport system. It should be stressed here that the cysteine transport system rapidly loses its
853
CYSTEINE TRANSPORT
Table 4. Effects of various chemicals on cysteine uptake* Concentration Chemical None EDTA HW4 Acetic acid KMnO, HgCh Thioglycol Ethanol (NH,),SO, L-Cystine D,L-Homocysteine Cysteic acid Cysteamine L-Glutathione L-Cystathionine L-Methionine DNP NaN, KCN
(PM)
100
1000 3130 3130 100 37 2600 3130 100 100 100 1000 1000 100 100 100 100 1000 100 1000 1000
Activity (nmol min-' OD6oo-')
Relative activity (YO) 100
0.78 f0.05 1.22k0.01 1.22 fO.01 0.38 f0.10 0.82 f0.09 0.45 f0.04 0.08 f0.03 0.43 f0.11 0.52 f0.00 0.93 f0.1 1 0.97 f0.12 0.67 f0.04 0.80 f0.12 0.90 f0.18 0.83 f0.07 0.78 f0.05 0.48 f0.03 0.2 1 k0.05 0.02 f0.00 0.13 f0.02 0.03 f0.00 0.42 0.08
155
155 49 104 57 10 55 66 119 123 85 102 115
106 100 62 28 2 17 4
53
*Strain NA22-10C (metl7) was used. Cysteine uptake was assayed in the presence of 100 PMcysteine and the indicated chemicals at the indicated concentration. DNP, Dinitrophenol.
.-C
E
x
4-
3-
T.
0
E
v
?o
2-
.= .-> c
v
0
o
x
.-v '5
0.8
854
B. O N 0 AND K. NAITO
a , t
2.5
%
0.0
I
0
1
I
7 4 - b
2 3 Time ( h r )
15
48
Figure 5. Derepression of the cysteine transport system. Strain IS66-4C (wild type) was grown in YPD liquid medium, harvested, washed twice with water, suspended in sulfur-free liquid medium, and incubated at 30°C with rotary shaking (60 rpm). After intervals, cysteine transport activity was assayed. The cells were treated with (0)or without ( 0 )50 pg/ml cycloheximide; cycloheximide was added to Y P D medium 15 min before the transfer and to sulfur-free medium to which the cells were transferred.
activity in buffer (Figure 1). From this observation, together with the effective inactivation of the system by metabolic inhibitors (Table 4), we conclude that the system depends on energy metabolism. We have found that the cysteine transport system is non-competitively inhibited by methionine (Figure 3). According to Gits and Grenson (1967), methionine transport is not inhibited by cysteine. Therefore, it appears as if S. cerevisiue takes up methionine preferentially over cysteine. As we have argued in the preceding paper (On0 et ul., 1991), S . cerevisiue appears to be programmed to utilize methionine preferentially if it is available from the environment. The present finding indicates that the regulation of transport systems operates under the same principle. It has been found that cysteine transport activity is low in prototrophic strains, which may be the reason why attention has not previously been drawn to the cysteine transport system. We have found that cysteine-dependent mutants are higher in cysteine uptake activity than the prototrophic strains. We have clearly shown that the cysteine transport system is repressed in the prototrophic strains and that derepression of the system takes place if the strains are incubated in medium lacking any sulfur source. The result that cysteine transport activity of the cysteine-dependent strains increased with increasing supply of organic sulfur sources is contradictory to our present argument. Our rationale at the moment is as follows. The cellular cysteine level required for repression of the cysteine uptake
system is much higher than that for cystathionine y-lyase. So that, under conditions where cystathionine y-lyase is repressed, the cysteine transport system remains derepressed. In such circumstances, increasing the supply of cysteine results in the increased biosynthesis of the cysteine transport system if cysteine is the limiting factor. That is, our speculation predicts that the cysteine transport system is rich in cysteine. Inhibition of the cysteine transport system by thioglycol (Table 4) favors the presence of SH-group(s) in the cysteine transport system. Strong inhibition by HgCl, (Table 4) may also be taken as another indication that the cysteine transport system contains the SH-group. However, there is a possibility that HgCl, inhibits some other biological functions such as energy metabolism, which then affects the cysteine transport activity. Anyway, it is important to know the amino acid composition of the cysteine transport system to support our hypothesis. ACKNOWLEDGEMENTS We thank D r S. Ohmori (Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan) for supply of cysteic acid. We thank Dr S. Shinoda (Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan) for his valuable suggestions during the work. We also thank the staff of the Tsushima Radioisotope Laboratory of Okayama University, where the radioisotope experiments were carried out. REFERENCES Gits, J. J. and Grenson, M. (1967). Multiplicity of the amino acid permeases in Saccharomyces cercvisiae. 111. Evidence for a specific methionine-transporting system. Biochim Biophys. Acta 135,507-5 16. Greasham, R. L. and Moat, A. G. (1973). Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae. J . Bacteriol. 115,975-98 1. Grenson, M., Hous, C . and Crabeel, M. (1970). Multiplicity of the amino acid permease in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J . Bacteriol. 103,770-777. Mortimer, R. (1987). Yeast Genetic Stock Center CUUlogue. Yeast Genetic Center, University of California, Berkeley, California. Naiki, N. and Iwata, M. (1962). Utilization of sulfur compounds by methionineless mutants of Saccharomycrs cerevisiae. Sci.Rep. Fuc. Lib. Arts Educ., Glfu Univ. 3, 70-75. (In Japanese with an English summary.)
CYSTEINE TRANSPORT
Ono. B., Suruga, T., Yamamoto, M., Yamamoto, S., Murata, K., Kimura, A,, Shinoda, S. and Ohmori, S. (1984). Cystathionine accumulation in Saccharomyces cerevisiae. J . Bacteriol. 158,86&865. Ono, B., Shirahige, Y., Nanjoh, A,, Andou, N., Ohue, H. and Ishino-Arao, Y. (1988). Cysteine biosynthesis in Saccharomyces cerevisiae: mutation that confers cystathionine (3-synthase deficiency. J . Bacteriol. 170, 5883-5889.
855 Ono, B., Naito, K., Shirahige, Y. and Yamamoto, M. (1991). Regulation ofcystathionine y-lyasein Saccharomyces cerevisiae. Yeast 7,843-848. Sherman, F., Fink, G. R. and Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Wickerham, L. J. (1956). A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeast. J. Bacteriol. 52,293-301.
