YEAST
VOL. 6:263-270
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GABA Transport in Saccharomyces cerevisiae JOYCE McKELVEY, RAJENDRA RAI AND TERRANCE G. COOPER* Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163, U S .A .
Received 7 September 1989; revised 26 December 1989
Gamma-aminobutyrate(GABA)accumulation in growingculturesof Saccharomyces cerevisiae was shown to occur by means of an active transport system that is inhibited by proton ionophores,azide, fluoride and arsenateions. Transport occurred maximally at pH 5.0 and exhibited apparent K , values of 12 ~ L and M 0.1 mM. Accumulated GABA did not efflux upon treatment with proton ionophores and exchanged with extracellular material only very slowly. However, release was complete upon treatment with nystatin. These observations raise the possibility that a major portion of intracellularGABA is sequesteredin the vacuole. The response of GABA uptake to growth on various nitrogen sources suggested that uptake may be subject to several types of regulation. KEYWORDS-GABA Transport.
INTRODUCTION Saccharomyces cerevisiae can utilize gammaaminobutyrate (GABA) by transaminating the gamma-amino group to 2-0x0-glutarate and degrading the resulting succinate semialdehyde to succinate (Pietruszko and Fowden, 1961). The GABA transaminase and succinic semialdehyde dehydrogenase activities required for these reactions have been detected in this and other yeasts (Jakoby and Scott, 1959; Scott and Jakoby, 1959). Mutants lacking these activities have also been recently reported (Ramos et al., 1985). There has been, however, no characterization of the transport system associated with GABA accumulation. Therefore, the purpose of this work was to define the characteristics of the GABA transport system. We found GABA uptake to involve high and low K,,, transport systems. Pre-accumulated GABA was unable to exit from the cell or exchange with extracellular ligand, a characteristic reported in the past for metabolites that are sequestered in the vacuole.
citrate (the pH was adjusted with HCI). Glucose, at a final concentration of O.6%, was used as sole carbon source. Whenever necessary, nutritional requirements of strains were satisfied by adding appropriate supplements to the medium. Nitrogen sources were provided at a final concentration of 0-1YOwith the exception of GABA, which was provided at a final concentration of 10 mM. Cell density measurements were made with a Klett Summerson colorimeter (500-570 nm band pass filter). One hundred Klett units is equivalent to approximately 3 x 10’ cells per ml of culture. Table 1. Strains Strain GC 210 GC 213 M970
M1473
METHODS
Genotype MATa lys2 MATa lysS MATa lys2 MATa lys5 MATa lys5 gab1 MATa Iys2 gab1
Strains and culture conditions
The strains used in this work are shown in Table 1. All are derivatives of strain E1278b. The medium used throughout was that of Wickerham (1946). The medium was buffered by addition of 1% sodium *Author to whom correspondence should be sent 0749-503X/90/03026348 $05.00 0 1990 by John Wiley & Sons Ltd
Resting cell cultures were prepared as follows. Cells were grown in buffered glucose, proline medium to a cell density of 43 Klett units. The cells were then harvested by filtration and washed in several volumes of nitrogen-free medium and resuspended in an equal volume (original) or prewarmed,
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Table 2. Growth of wild-type and gab1 mutant strains on various nitrogen sources
H
Doubling time (min) Nitrogen source Allantoin Urea Ammonia y-aminobutyrate Arginine Asparagine Glutamate Glutamine Ornithine Proline
Wild-type"
gabl
21 1 I99 166 213 191 183 I?? 187 448 181
312 234 173 NG' 171 I69 173 150 393 203
,'Strain M970. 'Strain M1473. 'No detectable growth after 48 h. All values were determined using Wickerham's minimal medium with glucose as sole carbon source. All values were normalized to those observed when proline was provided as sole nitrogen source. Nitrogen sources were provided at a final concentration of 0. I YOexcept y-aminobutyrate, which was provided at 10 mM.
pre-aerated medium that was devoid of nitrogen source. After incubation at 30°C for 19-20 h, the culture was used for the assay of GABA uptake. The cell density at this time was 134 Klett units. Isolation of mutant strains Strain GC-120 was grown to stationary phase in YEPD medium, harvested by centrifugation, washed, and then resuspended in 0.05 M-phosphate buffer (pH 8.0). The culture was mutagenized with 3% ethyl methane sulfonate for 60min at 30°C. After this treatment, the culture was harvested and suspended in a 6% sodium thiosulfate solution, and then washed with water. The method used to enrich for the desired mutant strain was that described by Snow (1966) and Fink (1970). Purification of the mutants, complementation tests and outcrossing were performed using standard genetic methods (Fink, 1970; Mortimer and Hawthorne, 1969). The gabl phenotype segregated 2 + : 2- in 37 tetrads, which is consistent with it being derived from a single locus alteration. Transfer of cellsfrom one medium to another In several experiments we had to transfer cells from one medium to another. This was done by
0
10
20
30
40
50
60
FRACTION NUMBER Figure 1. Recovery and analysis of accumulated gammaaminobutyrate (GABA) from S. cerevisiue. Strain MI473 was grown to a cell density of45 Klett units. ('HIGABA was added to the culture at a final concentration of 0.25 mM. After incubation for 40 min, the cells were harvested by filtration, washed with medium, a small sample was taken to determine total radioactive material accumulated and the remainder resuspended in 1 ml of 70% ethanol. The cells were permeabilized by vortexing. Following separation of particulate material from the supernatant by centrifugation, the supernatant was dried under vacuum and resuspended in a small amount of glass-distilled water. A sample of this extract was subjected to paper chromatography along with a sample of authentic GABA. The chromatogram was developed and processed as described in Methods. Within experimental error, all of the radioactivity observed to be accumulated within the cells prior to their permeabilization was loaded onto the chromatogram.
filtering the culture and suspending the cells in fresh, prewarmed, pre-aerated medium. All filtrations were performed with nitrocellulose filters (0.45 pm pore diameter, Milliport Corp.) and were completed in less than 15-20 s. The extent of cell loss during this procedure was determined with radioactively labelled cells and was found to be negligible. Assay of GABA uptake At zero time a 20 ml portion of the culture to be assayed was transferred to a flask containing 0.25 ~ M - [ ~ H ] G A BIncubation A. was carried out at 30°C in a shaking water bath under conditions that
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GABA TRANSPORT IN YEAST
RESULTS Development and verijication of a GABA transport assay system
MINUTES Figure 2. Time-dependent distribution of GABA between cells and medium. A culture of strain MI473 was grown to mid-log phase and starved overnight. The next morning (cell density, 134 Klett units). [)H]GABA (final concentration, 10 p ~ was ) added to 20 ml of this culture. Thereafter, at the times indicated, I ml samples were removed from the culture and the cells were separated from the culture medium by filtration. The amount ofradioactivity each pair of samples contained was then determined. The data are expressed as the amount of GABA present in 1 ml ofcells or medium.
were identical to those used for growth. At the desired times, 1.O ml samples were transferred from the flask to a nitrocellulose membrane filter. These filters were washed three times with 5 ml of medium containing 10 mhl-non-radioactive GABA. Washed filters were then placed in 5 ml of aqueous scintillation fluid (Aquasol, New England Nuclear Corp.) and the amount of radioactivity they contain was determined 16-24 h later. This period of time permitted any chemiluminescence to dissipate. Paper chromatography Compounds to be analysed were spotted on Whatman No. 541 filter paper. The chromatogram was developed after approximately 5.5 h with isopropanol : ammonium hydroxide (7 : 3) as solvent. After development, the paper was air dried in a fume hood and sprayed with ninhydrin for visual observation. For radioactivity determination, the paper was cut into 0.5 cm strips.
An accurate assay of GABA transport requires that its uptake be dissociated from metabolism. Therefore, we isolated a mutant strain of S. cerevisiae that was able to transport GABA, but was unable to metabolize it. The diploid strain (M1473) derived from this mutant grew normally on a variety of nitrogen sources, but failed to grow detectably when GABA was provided as sole nitrogen source (Table 2 ) . Following construction of the appropriate mutant strain for the transport experiments, it was important to demonstrate that GABA accumulated by the strain was not metabolized in any way. This was accomplished by incubating a culture with commercially prepared radioactive GABA and then analysing intracellularly accumulated material. Following incubation of strain M1437 with 0.25 ~ M - [ ~ H ] G A Bfor A 41 min, the cells were harvested by filtration, washed four times with 5 ml of wash solution, and the intracellular contents extracted with 70% ethanol. This extract was concentrated and chromatographed as described in Methods. As shown in Figure 1, all of the radioactive material recovered from the cells migrated in a single sharp peak with an R , value that was indistinguishable from that of authentic GABA (the bar at the top of the chromatogram indicates the area occupied by authentic GABA stained with ninhydrin reagent). These data suggested that conditions for an unambiguous assay of intracellular GABA accumulation had been established. Accumulation of GABA against a concentration gradient A means of clearly distinguishing active transport from facilitated diffusion is whether or not the solute can be accumulated against a concentration gradient. To test for GABA accumulation, a culture of strain M 1473was grown up to mid-log phase and starved overnight (20 h). The starved cells, at a cell density of 134 Klett units, were then incubated with radioactive GABA. Samples were removed at timed intervals and cells were separated from the medium. The amount of radioactive GABA that each contained was then determined. As shown in Figure 2, GABA accumulated in a culture of strain M1473 for about 50 min before reaching a plateau. GABA correspondingly disappeared from the medium. The cell sample removed after 100min of incubation
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J. McKELWY, R. RAI AND T. G. COOPER
180 160
Lu
140
40
20
0
20
40
60
80
MINUTES
0
20
40
60
80
MINUTES
Figure 3. Efflux and exchange of pre-accumulated GABA. A culture of strain M 1473 was prepared as described in Figure 1. At zero time, the culture was divided into two portions and incubated with 0.25 ~M-[’H]GABA. Samples were removed from each culture for assay at the times indicated. After 15 min of incubation, one culture (panel A) was harvested and resuspended in fresh medium devoid of GABA. The second culture (panel B) was harvested and resuspended in fresh medium containing 10 mM-non-radioactive GABA. Thereafter, samples were removed from each culture and the amount of intracellular, [’HIGABA they contained was determined as described in Methods.
contained 395nmol of GABA compared to 18 remaining in the medium. Given the small volume of cells assayed (4 x lo7 cells/ml and approximately 43 pm3/cell [Cooper et al., 1979; Cooper and Sumrada, 1975; Sumrada and Cooper, 1977)], GABA was concentrated several thousand-fold within the cells.
EBux and exchange of preloaded GABA The high intracellular concentration of GABA achieved in the above experiment prompted us to determine whether or not pre-accumulated GABA could be easily removed from cells. This was done
by permitting cells to accumulate GABA (0.25 mM) for 15 min and then resuspending them in fresh, prewarmed, pre-aerated medium devoid of GABA. Thereafter, we sampled the culture and measured the amount of radioactivity the cell samples contained. As shown in Figure 3A, no detectable efflux of pre-accumulated GABA was observed during the 80-min incubation period. The GABA uptake system was, however, capable of a limited exchange reaction. This was shown by preloading cells with [3H]GABA and then adding a large excess of nonradioactive GABA. Asshownin Figure 3B, addition of the non-radioactive GABA resulted in a slow loss
267
GABA TRANSPORT IN YEAST
0.07 0.06 -
0.05 -
0.04 0.03 -
0.02 -
’ ’ /
0.01’-
/
J
20
’
/ 10
10
20
30 1 [y-AMINOBUTYRATE]
40
Figure 4. Response of the rate of GABA accumulation to increasing extracellular concentrations of GABA. A culture of strain M1473 was grown to a cell density of 45 Klett units in buffered glucose proline medium. Samples (1 ml) of the culture were transferred to prewarmed reaction flasks containing varying concentrations of radioactive GABA. After 15 min of incubation, the sample was removed and the amount of radioactive GABA accumulated determined.
Table 3. Effect of various inhibitors of energy metabolism on uptake of y-aminobutyrate into strain M 1473
Inhibitor None 1 mM-Dinitrophenol 1 mM-Potassium cyanide 5 mw-sodium fluoride 1 mM-Sodium azide 5 mM-sodium arsenate 0.1 mM-CCP
Rate of uptake YOactivity (nmol/l2 min) remaining 52.41 0.22 32.58 2.06 3.97 6.03 1.1 1
100
0.4 62.2 3.9 7.6 11.5 2.1
A culture of strain M 1473 was grown in glucose proline medium to a cell density of 54 Klett units. 2 ml portions of the culture were transferred to a flask containing either 1 mM-potassium cyanide, 5 mu-sodium arsenate, 5 mM-sodium azide, 5 mMsodium fluoride, 1 mM-sodium azide, 0.1 mM-carbonyl cyanidern-chorophenyl hydrazone (CCCP), 1 mM-DNP or no inhibitor, respectively. After 10 min of incubation, 0.25 ~M-[’H]GABA was added to each flask. After 12 min of incubation, 1 ml samples were removed from each flask at 2-min intervals and processed as described in the text.
of intracellular radioactivity during the 80 min the cultures were monitored. Response of GABA uptake rate to increasing concentrations of solufe
Figure 4 depicts a Lineweaver-Burk plot of the initial rate of GABA uptake observed at increasing external GABA concentrations. A clearly non-linear response was found, indicating the probable existence of high the low K, systems. The apparent K,,, values of the high and low K, systems were extrapolated to be approximately 0.1 mM and 0.012 mM, respectively. This is in the same range of values observed for the allantoin and allantoate transport systems (Sumrada and Cooper, 1977; Turoscy and Cooper, 1979). p H optimum of GABA uptake
GABA uptake occurred over a rather broad pH range with a maximum around pH 5-O(Figure5). This compares with optima of 5.0-5.5 and 5.75 for
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J. McKELVEY, R. RAI AND T. G. COOPER
Treatment with nystatin brought about a rapid release of previously accumulated GABA, as was observed with urea (Cooper and Sumrada, 1975), allantoin (Sumrada and Cooper, 1977),or oxalurate (Cooper et al., 1979) (Figure 6B). EfSect of various nitrogen sources on GABA uptake
To gather an initial insight into the regulation of GABA uptake, cultures of strain M1473 were grown in the presence of a variety of nitrogen sources. Samples were then taken for determination of initial rates of GABA uptake. As shown in Table 4, uptake was maximal when proline was provided as nitrogen source. Uptake in minimal glutamate or glutamate + proline media, on the other hand, was significantly lower. There was also significant uptake in cultures grown in threonine glutamate or threonine + valine media. Uptake was quite low for the remainder of the nitrogen sources tested.
+
2.0
3.0
5.0
4.0
6.0
7.0
PH Figure 5. Response of GABA uptake to varying pH. A culture of strain M1473 was grown to a cell density of 45 Klett units in glucose proline medium. Portions (4.5 ml) of the culture were added to reaction flasks containing 0.5 ml of 1 M-citrate of phosphate buffers of the desired pH. After 1 min of incubation at 30"C, 1.2ml aliquots were removed and assayed for GABA accumulation using the assay described in Methods and a final GABA concentration of 0.25 mM.
the transport of allantoin and allantoate, respectively (Sumrada and Cooper, 1977; Turoscy and Cooper, 1979). It is significantly higher than that observed for urea (3.0-3.5) (Cooper and Sumrada, 1975). Requirement of a driving force f o r uptake of GABA
To access the energy dependence of GABA uptake, we monitored the initial uptake rates in the presence of several compounds known to inhibit various steps of energy metabolism. As shown in Table 3, GABA uptake was significantly inhibited by the proton ionophores dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone, as well as by fluoride, azide and arsenate ions. Uptake was rather insensitive to inhibition by cyanide ion. Although energy was required to accumulate GABA, it did not appear to be required to maintain pre-accumulated GABA within the cell. Addition of dinitrophenol to cultures accumulating GABA (1 5 min) inhibited further accumulation, but did not result in any loss of radioactive material accumulated before addition of the inhibitor (Figure 6A).
DISCUSSION Experiments presented here demonstrate that GABA is transported into the cell by at least two energy-dependent transport systems with apparent K, values of 12 pm and 0.1 mM. Transport occurred over a broad pH range with a maximum at pH 5.0. Whether the two systems are both specific for GABA, as is the case for urea (Cooper and Sumrada, 1975), is not clear at this time. Previous studies of the transport systems of nitrogenous compounds permit dividing the systems into two categories. The first category is exemplified by urea transport. Intracellularly accumulated urea rapidly effluxes from the cell upon addition of dinitrophenol, and exhibits a rapid rate of exchange with extracellular material (Cooper and Sumrada, 1975). The second category is exemplified by allantoin accumulation. Intracellularly accumulated allantoin does not efflux from the cell upon addition of dinitrophenol, nor does it readily exchange with extracellular material (Sumrada and Cooper, 1977). However, treatment with nystatin results in rapid loss of allantoin from the cell. Similar observations have been made for a variety of amino acids such as arginine and histidine (Crabeel and Grenson, 1970; Greth et al., 1977; Kotyk and Rihova, 1972), and other metabolites such as allantoate (Turoscy Cooper, 1979). The efflux and exchange characteristics of accumulated metabolites usually correlate with their intracellular distribution (Sumrada and Cooper,
269
GABA TRANSPORT IN YEAST
MINUTES
MINUTES
Figure 6 . Effect of dinitrophenol and nystatin on the ability of S. cerevisiae cells to maintain pre-accumulated intracellular GABA within the cell. A culture ofstrain M 1473 was grown in glucose proline medium to a cell density of40 Klett units. Two 20 ml portions were transferred to flasks containing sufficent GABA to yield a final concentration of 0 2 5 mM, and samples were removed for assay of accumulated material at the times indicated. At 16 min ten samples of these cultures were transferred to a second flask containing either dinitrophenol (DNP, 1 mM final concentration) or nystatin (18 pg/ml). Samples were removed at the indicated times and the amount of radioactive GABA they contained was determined as before.
1978; Zacharski and Cooper, 1978). The majority of intracellular allantoin, arginine, histidine and allantoate has been shown to be sequestered within the vacuole (Sumrada and Cooper, 1978; Zacharski and Cooper, 1978). Urea, in contrast, has been shown to be situated within the cytoplasm; little if any can be demonstrated within the vacuole (Sumrada and Cooper, 1978; Zacharski and Cooper. 1978). This correlation is consistent with the suggestion that intracellular GABA is divided into two pools: a small cytoplasmic pool that can be degraded and a much larger, sequestered vacuolar pool. Alternatively, there is an unlikely possibility that GABA transport is a unidirectional process (Crabeel and Grenson, 1970; Grenson, 1973; Kotyk and Rihova, 1972; Surdin et al., 1965). The intracellular distribution of GABA will be important as the regulation of its uptake and metabolism are studied in more detail.
The response of GABA uptake to growth on various amino acids suggests that the regulation of the system may be quite complex. For example, the very low level of transport observed when glutamine was provided as nitrogen source is consistent with uptake being sensitive to nitrogen catabolite repression. However, the fact that allantoin and arginine support less uptake than did glutamate would not support that hypothesis. The basis of the observed stimulatory effect of threonine in the presence of valine or glutamate is similarly obscure. These questions can only be addressed when the uptake systems have been isolated from one another genetically OT the pertinent genes are cloned. ACKNOWLEDGEMENTS The authors wish to thank the UT yeast group for their efforts in reading this manuscript and offering
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Table 4. Effects of various nitrogen sources on the uptake of y-aminobutyrate Nitrogen source Proline Glutamate Proline glutamate Glutamine Threonine Glutamate + threonine Valine Threonine + valine Allantoin Allantoate Ureidoglycollate Urea Arginine Ornithine Alanine Serine Asparagine Aspartate
+
Rate of uptake (nmol/min) 4.48 0.87 1.48 0.01
0.58 3.34 0.56 1.47 0.18 0.40 0.88 0.58 0.10 0.55 0.06 0.05 0.02 0.02
Growth conditions were described in Table 2. The concentration of [3H]y-aminobutyrate provided for the assay of uptake was 0.25 mM. Strain M1473 was used for this experiment.
suggestions for its improvement. This work was supported by United States Public Health Service
Grants GM-35642, and GM-35536.
REFERENCES Cooper, T. G., McKelvey, J. and Sumrada, R. (1979). Oxalurate transport in Saccharomyces cerevisiae. J . Bacteriol. 139,917-923. Cooper, T. G. and Sumrada, R. (1975). Urea transport in Saccharomyces cerevisiae. J. Bacteriol. 121,57 1-576. Crabeel, M. and Grenson, M. (1970). Regulation of histidine uptake by specific feedback inhibition of two histidine permeases in Saccharomyces cerevisiae. Eur. J. Biochem. 14,197-204. Fink, G. R. (1970). The biochemical genetics of yeast. Methods of Enzymology 17A, 59-78. Grenson, M. (1973). Specificity and regulation of the uptake and retention of amino acids and pyrimidines in
yeast. In Vanek, Z., Hostalek, Z. and Cudlin, J. (Eds), Genetics of Industrial Microorganisms. Academia, Praque, Czechoslovakia, pp. 179-193. Greth, M. L., Chevallier, M. R. and Lacroute, F. (1977). Ureidosuccinic acid permeation in Saccharomyces cerevisiae. Biochim. Biophys. Acta 465, 138-1 5 1. Jakoby, W. B. and Scott, E. M. (1959). Aldehyde oxidation. 111. Succinic semialdehyde dehydrogenase. J. Biol. Chem. 234,937-940. Kotyk, A. and Rihova, L. (1972). Transport of alphaamino isobutyric acid in Saccharomyces cerevisiae. Biochim. Biophys. Acta 288,380-389. Mortimer, R. K. and Hawthorne, D. C. (1969). Yeast genetics. In Rose, A. H. and Harrison, J. S. (Eds), The Yeasts, vol. 1. Academic Press, New York, pp. 385- 460. Pietruszko, R. and Fowden, L. (1961). Gammaaminobutyric acid metabolism in plants: Part 1. Metabolism in yeasts. Ann. Bot. (Lond.) 25,491-511. Ramos, F., El Guezzar, M., Grenson, M. and Wiame, J. M. (1985). Mutations affecting the enzymes involved in the utilization of 4-aminobutyric acid as nitrogen source by the yeast Saccharomyces cerevisiae. Eur. J . Biochem. 149,401-404. Scott, E. M. and Jakoby, W. B. (1959). Soluble gamma amino butyric glutamic transaminase from Pseudomonasjuorescens. J . Biol. Chem. 234,932-936. Snow, R. (1966). An enrichment method for auxotrophic mutants using the antiobiotic nystatin. Nature 211, 206-207. Sumrada, R. and Cooper, T. G. (1977). Allantoin transport in Saccharomyces cerevisiae. J. Bacteriol. 131, 839-847. Sumrada, R. and Cooper, T. G. (1978). Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae. J. Bacterioi. 136,234-246. Surdin, Y., Sly, W., Sire, J., Bordes, A. M. and RobichonSzulmajster, J. (1965). Properties et controle genetique du systeme d’accumulation des acides amines ches Saccharomyces cerevisiae. Biochim. Biophys. Actu 107, 546-566. Turoscy, V. and Cooper, T. G. (1979). Allantoate transport in Saccharomyces cerevisiae. J . Bacteriol. 140, 97 1-979. Wickerham, L. J. (1946). A critical evaluation of the nitrogen assimulation tests commonly used in the classification of yeasts. J. Bacteriol. 52,293-301. Zacharski, C. A. and Cooper, T. G. (1978). Metabolite compartmentation in Saccharomyces cerevisiae. J . Bacteriol. 135,490-497.
VOL. 6:263-270
(1 990)
GABA Transport in Saccharomyces cerevisiae JOYCE McKELVEY, RAJENDRA RAI AND TERRANCE G. COOPER* Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163, U S .A .
Received 7 September 1989; revised 26 December 1989
Gamma-aminobutyrate(GABA)accumulation in growingculturesof Saccharomyces cerevisiae was shown to occur by means of an active transport system that is inhibited by proton ionophores,azide, fluoride and arsenateions. Transport occurred maximally at pH 5.0 and exhibited apparent K , values of 12 ~ L and M 0.1 mM. Accumulated GABA did not efflux upon treatment with proton ionophores and exchanged with extracellular material only very slowly. However, release was complete upon treatment with nystatin. These observations raise the possibility that a major portion of intracellularGABA is sequesteredin the vacuole. The response of GABA uptake to growth on various nitrogen sources suggested that uptake may be subject to several types of regulation. KEYWORDS-GABA Transport.
INTRODUCTION Saccharomyces cerevisiae can utilize gammaaminobutyrate (GABA) by transaminating the gamma-amino group to 2-0x0-glutarate and degrading the resulting succinate semialdehyde to succinate (Pietruszko and Fowden, 1961). The GABA transaminase and succinic semialdehyde dehydrogenase activities required for these reactions have been detected in this and other yeasts (Jakoby and Scott, 1959; Scott and Jakoby, 1959). Mutants lacking these activities have also been recently reported (Ramos et al., 1985). There has been, however, no characterization of the transport system associated with GABA accumulation. Therefore, the purpose of this work was to define the characteristics of the GABA transport system. We found GABA uptake to involve high and low K,,, transport systems. Pre-accumulated GABA was unable to exit from the cell or exchange with extracellular ligand, a characteristic reported in the past for metabolites that are sequestered in the vacuole.
citrate (the pH was adjusted with HCI). Glucose, at a final concentration of O.6%, was used as sole carbon source. Whenever necessary, nutritional requirements of strains were satisfied by adding appropriate supplements to the medium. Nitrogen sources were provided at a final concentration of 0-1YOwith the exception of GABA, which was provided at a final concentration of 10 mM. Cell density measurements were made with a Klett Summerson colorimeter (500-570 nm band pass filter). One hundred Klett units is equivalent to approximately 3 x 10’ cells per ml of culture. Table 1. Strains Strain GC 210 GC 213 M970
M1473
METHODS
Genotype MATa lys2 MATa lysS MATa lys2 MATa lys5 MATa lys5 gab1 MATa Iys2 gab1
Strains and culture conditions
The strains used in this work are shown in Table 1. All are derivatives of strain E1278b. The medium used throughout was that of Wickerham (1946). The medium was buffered by addition of 1% sodium *Author to whom correspondence should be sent 0749-503X/90/03026348 $05.00 0 1990 by John Wiley & Sons Ltd
Resting cell cultures were prepared as follows. Cells were grown in buffered glucose, proline medium to a cell density of 43 Klett units. The cells were then harvested by filtration and washed in several volumes of nitrogen-free medium and resuspended in an equal volume (original) or prewarmed,
264
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Table 2. Growth of wild-type and gab1 mutant strains on various nitrogen sources
H
Doubling time (min) Nitrogen source Allantoin Urea Ammonia y-aminobutyrate Arginine Asparagine Glutamate Glutamine Ornithine Proline
Wild-type"
gabl
21 1 I99 166 213 191 183 I?? 187 448 181
312 234 173 NG' 171 I69 173 150 393 203
,'Strain M970. 'Strain M1473. 'No detectable growth after 48 h. All values were determined using Wickerham's minimal medium with glucose as sole carbon source. All values were normalized to those observed when proline was provided as sole nitrogen source. Nitrogen sources were provided at a final concentration of 0. I YOexcept y-aminobutyrate, which was provided at 10 mM.
pre-aerated medium that was devoid of nitrogen source. After incubation at 30°C for 19-20 h, the culture was used for the assay of GABA uptake. The cell density at this time was 134 Klett units. Isolation of mutant strains Strain GC-120 was grown to stationary phase in YEPD medium, harvested by centrifugation, washed, and then resuspended in 0.05 M-phosphate buffer (pH 8.0). The culture was mutagenized with 3% ethyl methane sulfonate for 60min at 30°C. After this treatment, the culture was harvested and suspended in a 6% sodium thiosulfate solution, and then washed with water. The method used to enrich for the desired mutant strain was that described by Snow (1966) and Fink (1970). Purification of the mutants, complementation tests and outcrossing were performed using standard genetic methods (Fink, 1970; Mortimer and Hawthorne, 1969). The gabl phenotype segregated 2 + : 2- in 37 tetrads, which is consistent with it being derived from a single locus alteration. Transfer of cellsfrom one medium to another In several experiments we had to transfer cells from one medium to another. This was done by
0
10
20
30
40
50
60
FRACTION NUMBER Figure 1. Recovery and analysis of accumulated gammaaminobutyrate (GABA) from S. cerevisiue. Strain MI473 was grown to a cell density of45 Klett units. ('HIGABA was added to the culture at a final concentration of 0.25 mM. After incubation for 40 min, the cells were harvested by filtration, washed with medium, a small sample was taken to determine total radioactive material accumulated and the remainder resuspended in 1 ml of 70% ethanol. The cells were permeabilized by vortexing. Following separation of particulate material from the supernatant by centrifugation, the supernatant was dried under vacuum and resuspended in a small amount of glass-distilled water. A sample of this extract was subjected to paper chromatography along with a sample of authentic GABA. The chromatogram was developed and processed as described in Methods. Within experimental error, all of the radioactivity observed to be accumulated within the cells prior to their permeabilization was loaded onto the chromatogram.
filtering the culture and suspending the cells in fresh, prewarmed, pre-aerated medium. All filtrations were performed with nitrocellulose filters (0.45 pm pore diameter, Milliport Corp.) and were completed in less than 15-20 s. The extent of cell loss during this procedure was determined with radioactively labelled cells and was found to be negligible. Assay of GABA uptake At zero time a 20 ml portion of the culture to be assayed was transferred to a flask containing 0.25 ~ M - [ ~ H ] G A BIncubation A. was carried out at 30°C in a shaking water bath under conditions that
265
GABA TRANSPORT IN YEAST
RESULTS Development and verijication of a GABA transport assay system
MINUTES Figure 2. Time-dependent distribution of GABA between cells and medium. A culture of strain MI473 was grown to mid-log phase and starved overnight. The next morning (cell density, 134 Klett units). [)H]GABA (final concentration, 10 p ~ was ) added to 20 ml of this culture. Thereafter, at the times indicated, I ml samples were removed from the culture and the cells were separated from the culture medium by filtration. The amount ofradioactivity each pair of samples contained was then determined. The data are expressed as the amount of GABA present in 1 ml ofcells or medium.
were identical to those used for growth. At the desired times, 1.O ml samples were transferred from the flask to a nitrocellulose membrane filter. These filters were washed three times with 5 ml of medium containing 10 mhl-non-radioactive GABA. Washed filters were then placed in 5 ml of aqueous scintillation fluid (Aquasol, New England Nuclear Corp.) and the amount of radioactivity they contain was determined 16-24 h later. This period of time permitted any chemiluminescence to dissipate. Paper chromatography Compounds to be analysed were spotted on Whatman No. 541 filter paper. The chromatogram was developed after approximately 5.5 h with isopropanol : ammonium hydroxide (7 : 3) as solvent. After development, the paper was air dried in a fume hood and sprayed with ninhydrin for visual observation. For radioactivity determination, the paper was cut into 0.5 cm strips.
An accurate assay of GABA transport requires that its uptake be dissociated from metabolism. Therefore, we isolated a mutant strain of S. cerevisiae that was able to transport GABA, but was unable to metabolize it. The diploid strain (M1473) derived from this mutant grew normally on a variety of nitrogen sources, but failed to grow detectably when GABA was provided as sole nitrogen source (Table 2 ) . Following construction of the appropriate mutant strain for the transport experiments, it was important to demonstrate that GABA accumulated by the strain was not metabolized in any way. This was accomplished by incubating a culture with commercially prepared radioactive GABA and then analysing intracellularly accumulated material. Following incubation of strain M1437 with 0.25 ~ M - [ ~ H ] G A Bfor A 41 min, the cells were harvested by filtration, washed four times with 5 ml of wash solution, and the intracellular contents extracted with 70% ethanol. This extract was concentrated and chromatographed as described in Methods. As shown in Figure 1, all of the radioactive material recovered from the cells migrated in a single sharp peak with an R , value that was indistinguishable from that of authentic GABA (the bar at the top of the chromatogram indicates the area occupied by authentic GABA stained with ninhydrin reagent). These data suggested that conditions for an unambiguous assay of intracellular GABA accumulation had been established. Accumulation of GABA against a concentration gradient A means of clearly distinguishing active transport from facilitated diffusion is whether or not the solute can be accumulated against a concentration gradient. To test for GABA accumulation, a culture of strain M 1473was grown up to mid-log phase and starved overnight (20 h). The starved cells, at a cell density of 134 Klett units, were then incubated with radioactive GABA. Samples were removed at timed intervals and cells were separated from the medium. The amount of radioactive GABA that each contained was then determined. As shown in Figure 2, GABA accumulated in a culture of strain M1473 for about 50 min before reaching a plateau. GABA correspondingly disappeared from the medium. The cell sample removed after 100min of incubation
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180 160
Lu
140
40
20
0
20
40
60
80
MINUTES
0
20
40
60
80
MINUTES
Figure 3. Efflux and exchange of pre-accumulated GABA. A culture of strain M 1473 was prepared as described in Figure 1. At zero time, the culture was divided into two portions and incubated with 0.25 ~M-[’H]GABA. Samples were removed from each culture for assay at the times indicated. After 15 min of incubation, one culture (panel A) was harvested and resuspended in fresh medium devoid of GABA. The second culture (panel B) was harvested and resuspended in fresh medium containing 10 mM-non-radioactive GABA. Thereafter, samples were removed from each culture and the amount of intracellular, [’HIGABA they contained was determined as described in Methods.
contained 395nmol of GABA compared to 18 remaining in the medium. Given the small volume of cells assayed (4 x lo7 cells/ml and approximately 43 pm3/cell [Cooper et al., 1979; Cooper and Sumrada, 1975; Sumrada and Cooper, 1977)], GABA was concentrated several thousand-fold within the cells.
EBux and exchange of preloaded GABA The high intracellular concentration of GABA achieved in the above experiment prompted us to determine whether or not pre-accumulated GABA could be easily removed from cells. This was done
by permitting cells to accumulate GABA (0.25 mM) for 15 min and then resuspending them in fresh, prewarmed, pre-aerated medium devoid of GABA. Thereafter, we sampled the culture and measured the amount of radioactivity the cell samples contained. As shown in Figure 3A, no detectable efflux of pre-accumulated GABA was observed during the 80-min incubation period. The GABA uptake system was, however, capable of a limited exchange reaction. This was shown by preloading cells with [3H]GABA and then adding a large excess of nonradioactive GABA. Asshownin Figure 3B, addition of the non-radioactive GABA resulted in a slow loss
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GABA TRANSPORT IN YEAST
0.07 0.06 -
0.05 -
0.04 0.03 -
0.02 -
’ ’ /
0.01’-
/
J
20
’
/ 10
10
20
30 1 [y-AMINOBUTYRATE]
40
Figure 4. Response of the rate of GABA accumulation to increasing extracellular concentrations of GABA. A culture of strain M1473 was grown to a cell density of 45 Klett units in buffered glucose proline medium. Samples (1 ml) of the culture were transferred to prewarmed reaction flasks containing varying concentrations of radioactive GABA. After 15 min of incubation, the sample was removed and the amount of radioactive GABA accumulated determined.
Table 3. Effect of various inhibitors of energy metabolism on uptake of y-aminobutyrate into strain M 1473
Inhibitor None 1 mM-Dinitrophenol 1 mM-Potassium cyanide 5 mw-sodium fluoride 1 mM-Sodium azide 5 mM-sodium arsenate 0.1 mM-CCP
Rate of uptake YOactivity (nmol/l2 min) remaining 52.41 0.22 32.58 2.06 3.97 6.03 1.1 1
100
0.4 62.2 3.9 7.6 11.5 2.1
A culture of strain M 1473 was grown in glucose proline medium to a cell density of 54 Klett units. 2 ml portions of the culture were transferred to a flask containing either 1 mM-potassium cyanide, 5 mu-sodium arsenate, 5 mM-sodium azide, 5 mMsodium fluoride, 1 mM-sodium azide, 0.1 mM-carbonyl cyanidern-chorophenyl hydrazone (CCCP), 1 mM-DNP or no inhibitor, respectively. After 10 min of incubation, 0.25 ~M-[’H]GABA was added to each flask. After 12 min of incubation, 1 ml samples were removed from each flask at 2-min intervals and processed as described in the text.
of intracellular radioactivity during the 80 min the cultures were monitored. Response of GABA uptake rate to increasing concentrations of solufe
Figure 4 depicts a Lineweaver-Burk plot of the initial rate of GABA uptake observed at increasing external GABA concentrations. A clearly non-linear response was found, indicating the probable existence of high the low K, systems. The apparent K,,, values of the high and low K, systems were extrapolated to be approximately 0.1 mM and 0.012 mM, respectively. This is in the same range of values observed for the allantoin and allantoate transport systems (Sumrada and Cooper, 1977; Turoscy and Cooper, 1979). p H optimum of GABA uptake
GABA uptake occurred over a rather broad pH range with a maximum around pH 5-O(Figure5). This compares with optima of 5.0-5.5 and 5.75 for
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J. McKELVEY, R. RAI AND T. G. COOPER
Treatment with nystatin brought about a rapid release of previously accumulated GABA, as was observed with urea (Cooper and Sumrada, 1975), allantoin (Sumrada and Cooper, 1977),or oxalurate (Cooper et al., 1979) (Figure 6B). EfSect of various nitrogen sources on GABA uptake
To gather an initial insight into the regulation of GABA uptake, cultures of strain M1473 were grown in the presence of a variety of nitrogen sources. Samples were then taken for determination of initial rates of GABA uptake. As shown in Table 4, uptake was maximal when proline was provided as nitrogen source. Uptake in minimal glutamate or glutamate + proline media, on the other hand, was significantly lower. There was also significant uptake in cultures grown in threonine glutamate or threonine + valine media. Uptake was quite low for the remainder of the nitrogen sources tested.
+
2.0
3.0
5.0
4.0
6.0
7.0
PH Figure 5. Response of GABA uptake to varying pH. A culture of strain M1473 was grown to a cell density of 45 Klett units in glucose proline medium. Portions (4.5 ml) of the culture were added to reaction flasks containing 0.5 ml of 1 M-citrate of phosphate buffers of the desired pH. After 1 min of incubation at 30"C, 1.2ml aliquots were removed and assayed for GABA accumulation using the assay described in Methods and a final GABA concentration of 0.25 mM.
the transport of allantoin and allantoate, respectively (Sumrada and Cooper, 1977; Turoscy and Cooper, 1979). It is significantly higher than that observed for urea (3.0-3.5) (Cooper and Sumrada, 1975). Requirement of a driving force f o r uptake of GABA
To access the energy dependence of GABA uptake, we monitored the initial uptake rates in the presence of several compounds known to inhibit various steps of energy metabolism. As shown in Table 3, GABA uptake was significantly inhibited by the proton ionophores dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone, as well as by fluoride, azide and arsenate ions. Uptake was rather insensitive to inhibition by cyanide ion. Although energy was required to accumulate GABA, it did not appear to be required to maintain pre-accumulated GABA within the cell. Addition of dinitrophenol to cultures accumulating GABA (1 5 min) inhibited further accumulation, but did not result in any loss of radioactive material accumulated before addition of the inhibitor (Figure 6A).
DISCUSSION Experiments presented here demonstrate that GABA is transported into the cell by at least two energy-dependent transport systems with apparent K, values of 12 pm and 0.1 mM. Transport occurred over a broad pH range with a maximum at pH 5.0. Whether the two systems are both specific for GABA, as is the case for urea (Cooper and Sumrada, 1975), is not clear at this time. Previous studies of the transport systems of nitrogenous compounds permit dividing the systems into two categories. The first category is exemplified by urea transport. Intracellularly accumulated urea rapidly effluxes from the cell upon addition of dinitrophenol, and exhibits a rapid rate of exchange with extracellular material (Cooper and Sumrada, 1975). The second category is exemplified by allantoin accumulation. Intracellularly accumulated allantoin does not efflux from the cell upon addition of dinitrophenol, nor does it readily exchange with extracellular material (Sumrada and Cooper, 1977). However, treatment with nystatin results in rapid loss of allantoin from the cell. Similar observations have been made for a variety of amino acids such as arginine and histidine (Crabeel and Grenson, 1970; Greth et al., 1977; Kotyk and Rihova, 1972), and other metabolites such as allantoate (Turoscy Cooper, 1979). The efflux and exchange characteristics of accumulated metabolites usually correlate with their intracellular distribution (Sumrada and Cooper,
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GABA TRANSPORT IN YEAST
MINUTES
MINUTES
Figure 6 . Effect of dinitrophenol and nystatin on the ability of S. cerevisiae cells to maintain pre-accumulated intracellular GABA within the cell. A culture ofstrain M 1473 was grown in glucose proline medium to a cell density of40 Klett units. Two 20 ml portions were transferred to flasks containing sufficent GABA to yield a final concentration of 0 2 5 mM, and samples were removed for assay of accumulated material at the times indicated. At 16 min ten samples of these cultures were transferred to a second flask containing either dinitrophenol (DNP, 1 mM final concentration) or nystatin (18 pg/ml). Samples were removed at the indicated times and the amount of radioactive GABA they contained was determined as before.
1978; Zacharski and Cooper, 1978). The majority of intracellular allantoin, arginine, histidine and allantoate has been shown to be sequestered within the vacuole (Sumrada and Cooper, 1978; Zacharski and Cooper, 1978). Urea, in contrast, has been shown to be situated within the cytoplasm; little if any can be demonstrated within the vacuole (Sumrada and Cooper, 1978; Zacharski and Cooper. 1978). This correlation is consistent with the suggestion that intracellular GABA is divided into two pools: a small cytoplasmic pool that can be degraded and a much larger, sequestered vacuolar pool. Alternatively, there is an unlikely possibility that GABA transport is a unidirectional process (Crabeel and Grenson, 1970; Grenson, 1973; Kotyk and Rihova, 1972; Surdin et al., 1965). The intracellular distribution of GABA will be important as the regulation of its uptake and metabolism are studied in more detail.
The response of GABA uptake to growth on various amino acids suggests that the regulation of the system may be quite complex. For example, the very low level of transport observed when glutamine was provided as nitrogen source is consistent with uptake being sensitive to nitrogen catabolite repression. However, the fact that allantoin and arginine support less uptake than did glutamate would not support that hypothesis. The basis of the observed stimulatory effect of threonine in the presence of valine or glutamate is similarly obscure. These questions can only be addressed when the uptake systems have been isolated from one another genetically OT the pertinent genes are cloned. ACKNOWLEDGEMENTS The authors wish to thank the UT yeast group for their efforts in reading this manuscript and offering
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Table 4. Effects of various nitrogen sources on the uptake of y-aminobutyrate Nitrogen source Proline Glutamate Proline glutamate Glutamine Threonine Glutamate + threonine Valine Threonine + valine Allantoin Allantoate Ureidoglycollate Urea Arginine Ornithine Alanine Serine Asparagine Aspartate
+
Rate of uptake (nmol/min) 4.48 0.87 1.48 0.01
0.58 3.34 0.56 1.47 0.18 0.40 0.88 0.58 0.10 0.55 0.06 0.05 0.02 0.02
Growth conditions were described in Table 2. The concentration of [3H]y-aminobutyrate provided for the assay of uptake was 0.25 mM. Strain M1473 was used for this experiment.
suggestions for its improvement. This work was supported by United States Public Health Service
Grants GM-35642, and GM-35536.
REFERENCES Cooper, T. G., McKelvey, J. and Sumrada, R. (1979). Oxalurate transport in Saccharomyces cerevisiae. J . Bacteriol. 139,917-923. Cooper, T. G. and Sumrada, R. (1975). Urea transport in Saccharomyces cerevisiae. J. Bacteriol. 121,57 1-576. Crabeel, M. and Grenson, M. (1970). Regulation of histidine uptake by specific feedback inhibition of two histidine permeases in Saccharomyces cerevisiae. Eur. J. Biochem. 14,197-204. Fink, G. R. (1970). The biochemical genetics of yeast. Methods of Enzymology 17A, 59-78. Grenson, M. (1973). Specificity and regulation of the uptake and retention of amino acids and pyrimidines in
yeast. In Vanek, Z., Hostalek, Z. and Cudlin, J. (Eds), Genetics of Industrial Microorganisms. Academia, Praque, Czechoslovakia, pp. 179-193. Greth, M. L., Chevallier, M. R. and Lacroute, F. (1977). Ureidosuccinic acid permeation in Saccharomyces cerevisiae. Biochim. Biophys. Acta 465, 138-1 5 1. Jakoby, W. B. and Scott, E. M. (1959). Aldehyde oxidation. 111. Succinic semialdehyde dehydrogenase. J. Biol. Chem. 234,937-940. Kotyk, A. and Rihova, L. (1972). Transport of alphaamino isobutyric acid in Saccharomyces cerevisiae. Biochim. Biophys. Acta 288,380-389. Mortimer, R. K. and Hawthorne, D. C. (1969). Yeast genetics. In Rose, A. H. and Harrison, J. S. (Eds), The Yeasts, vol. 1. Academic Press, New York, pp. 385- 460. Pietruszko, R. and Fowden, L. (1961). Gammaaminobutyric acid metabolism in plants: Part 1. Metabolism in yeasts. Ann. Bot. (Lond.) 25,491-511. Ramos, F., El Guezzar, M., Grenson, M. and Wiame, J. M. (1985). Mutations affecting the enzymes involved in the utilization of 4-aminobutyric acid as nitrogen source by the yeast Saccharomyces cerevisiae. Eur. J . Biochem. 149,401-404. Scott, E. M. and Jakoby, W. B. (1959). Soluble gamma amino butyric glutamic transaminase from Pseudomonasjuorescens. J . Biol. Chem. 234,932-936. Snow, R. (1966). An enrichment method for auxotrophic mutants using the antiobiotic nystatin. Nature 211, 206-207. Sumrada, R. and Cooper, T. G. (1977). Allantoin transport in Saccharomyces cerevisiae. J. Bacteriol. 131, 839-847. Sumrada, R. and Cooper, T. G. (1978). Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae. J. Bacterioi. 136,234-246. Surdin, Y., Sly, W., Sire, J., Bordes, A. M. and RobichonSzulmajster, J. (1965). Properties et controle genetique du systeme d’accumulation des acides amines ches Saccharomyces cerevisiae. Biochim. Biophys. Actu 107, 546-566. Turoscy, V. and Cooper, T. G. (1979). Allantoate transport in Saccharomyces cerevisiae. J . Bacteriol. 140, 97 1-979. Wickerham, L. J. (1946). A critical evaluation of the nitrogen assimulation tests commonly used in the classification of yeasts. J. Bacteriol. 52,293-301. Zacharski, C. A. and Cooper, T. G. (1978). Metabolite compartmentation in Saccharomyces cerevisiae. J . Bacteriol. 135,490-497.
