Vol. 121, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 571-576 Copyright i 1975 American Society for Microbiology
Urea Transport in Saccharomyces cerevisiae TERRANCE G. COOPER* AND ROBERTA SUMRADA Department of Biochemistry-FAS, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 11 October 1974
Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 ,M, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion. Saccharomyces cerevisiae can utilize urea as sole nitrogen source by degrading it in two steps to ammonia and CO2 (10). The enzymes responsible for this degradation are urea carboxylase and allophanate hydrolase (11). Their production is contingent upon the presence of allophanic acid (2, 13) or the gratuitous inducer oxaluric acid (9) and is subject to nitrogen repression when cells are grown on readily utilized amino acids (1). Although there is reasonable understanding of the biochemistry, genetics, and physiology of the urea-degradative enzymes, there is no information concerning how urea is taken into the cell. Domnas reported (3) that during growth on allantoin the urea generated appears in the medium suggesting its exit from the cell, but no further data are given. In view of this deficiency we have investigated the mechanisms by which urea is accumulated and report that two modes of uptake are operative. MATERIALS AND METHODS Strains and media. Two strains defective in urea metabolism were used in this work. M-62 lacks urea carboxylase and M-64 lacks allophanate hydrolase activity (11). These strains were used in place of the
gm pore size) (Millipore Corp.) and unless indicated otherwise washed four times with 8 ml of cold medium containing 10 mM urea. Washed filters were placed in 5 ml of Aquasol (New England Nuclear Corp.) scintillation fluid and counted 16 to 24 h later. The incubation time in Aquasol was needed to allow the filters to become transparent. Failure to do this results in unevenly quenched samples and loss of assay precision. The report by Leder and Perry (Fed. Proc. 26:394) that washing with cold medium leads to a rapid loss of accumulated substrates prompted evaluation of our washing procedures. The losses reported by these investigators do not occur in Saccharomyces treated with cold medium (Table 1). Note also that only 8 ml of medium are required to completely remove remaining external urea; 32 ml was routinely used, however, as a precaution. Unless otherwise indicated, all data are expressed as amounts of radioactive urea observed per milliliter of the original culture.
RESULTS A urea carboxylase-minus strain of S. cerevisiae (M-62) cannot accumulate ["C lurea when grown on minimal ammonia medium (closed circles) (Fig. 1A). However, if oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, is present during growth this strain is capable of urea accumulation. A quite different behavior is observed when this, experiment is repeated using an allophanate hydrolase-minus strain (M-64). [TC ]urea is accumulated in this strain even in the absence of exogenous gratuitous inducer (Fig. 1B). To decide whether oxaluric acid is functioning here as a urea or allophanate analogue the experiment in Fig. 1A was repeated using arginine as sole nitrogen source in place of ammonia. This assures a high intracellular concentration of urea during cell growth. The presence of large quantities of urea within cells did not significantly increase their ability to accumulate urea from the medium.
wild-type strain because urea taken into wild-type cells is rapidly metabolized to CO2 which is then released into the medium (12). The medium used here was that described earlier (2). Except where indicated otherwise the nitrogen source was 0.1% ammonium sulfate. Urea uptake assay. For assay of urea uptake cultures were grown at 30 C to a cell density of 1.3 x 107 cells per ml (45 Klett units). At zero time an 8.5-ml portion of the culture was transferred to a flask containing ['IC lurea (specific activity, 4.7 ;Ci/4mol). Incubation was carried out at 30 C in a shaking water bath under conditions identical to those used during growth. At appropriate times 1.0-ml samples were transferred to nitrocellulose membrane filters (0.45 71
TABLE 1. Washing procedures for assay of urea uptake Temp (C)
Wash solutiona
Minimal medium Minimal medium + 10 mM urea Minimal medium
Minimal medium (8 ml) Minimal medium (16 ml) Minimal medium (24 ml) Minimal medium (34 ml) a Volume of wash solution wise indicated.
36 A
4 4
22 4 4 4 4 was
nmol of urea/ml of culture 2 min
16 min
0.77 0.74
3.21 3.34
0.79
3.21
4 min
16 min
0.65 0.61 0.57 0.61
1.28 1.26 1.21 1.22
32 ml unless other-
levels of urea accumulated in cells grown on ammonia and arginine likely results from isotope dilution which arises from catabolism of arginine to urea. These data closely parallel those observed in studies demonstrating the contingency of allantoin degradative enzyme production upon the presence of allophanic acid. If indeed these data reflect an ability to transport urea into cells it should be possible to "chase" radioactive urea out of the cells by addition of a large excess of nonradioactive urea to the culture. This was observed for both strains (Fig. 3). Addition of excess nonradioactive urea to cultures actively accumulating [4C ]urea resulted in abrupt loss of radioactivity from the cells. The half-life of this loss was 9.0
M-62 o
/
3.0 -+OXLU° 2.4
O
2.4
z Cn
2.0
ai. C,)
o,1.8
1_j
-j
-j
1.2_
AMMONIA + OXLU
2.8
w
/
--OXLU *
uj
J. BACTERIOL.
COOPER AND SUMRADA
572
w
0(.
w
1.6
04 /
ARGININE
+ OXLU
0
1.2
-C06
w
2 B
a a.
Z
85
0
4
8 6 M-64
10
12
14 C)
w 0 2C
0.8
0.4
AGNNE ALONE
w
2
4
6
8
10
12
MINUTES FIG. 2. Effect of intracellular urea upon production of the urea uptake system. Three cultures of strain M-62 were grown respectively on minimal ammonia medium plus oxaluric acid (0), minimal arginine medium plus oxaluric acid (a) and minimal arginine medium alone (U). At a cell density of 45 Klett units each culture was harvested by filtration and resuspended in fresh, prewarmed, pre-aerated, minimal ammonia medium. Those cultures previously grown 8 4 6 10 12 14 16 2 in the presence of oxaluric acid also received oxaluric MINUTES acid after resuspension in fresh medium. The three FIG. 1. Uptake of urea in urea carboxylase, M-62 cultures were incubated in this medium for 30 min to (A) and allophanate hydrolase, M-64 (B) minus permit urea within the cells to leave. The time strains of Saccharomyces. Cultures of each strain required for this to occur was determined from the were grown on ammonia minimal medium in the data shown in Fig. 4. At the end of this incubation presence (0) or absence (-) of 0.5 mM oxaluric acid. period (zero time in the figure) urea uptake was Urea uptake assays were performed as indicated in assayed as described in Materials and Methods. Materials and Methods. External radioactive urea Failure to remove urea from cells grown on arginine concentration was 0.36 mM. using these procedures greatly decreased their ability to accumulate added radioactive urea. This is due to a However, this (Fig. 2) ability was observed if steady state amount already being present within the oxaluric acid was present during growth. It cells at the time of radioactive urea addition to the should be noted that the difference between the medium. -J
0
4
2
0 ~~~~~~M-64
IL 60 O
0 ~~~~~~M-62
3.2
0 50
a _j
573
UREA TRANSPORT
VOL. 121, 1975
D a
r EXCESS
UREA _j40 40- /ADDED
w 2
w
.5 30 U,
w
2.4
1.6
0.8
20
8
16
24
32
40
48
56
64
MINUTES FIG. 3. Reversibility of urea uptake in Saccharomyces. The conditions of this experiment are identical to those used for the induced cultures of Fig. 1A. However, at 16.5 min nonradioactive urea was added to the assay mixture to a final concentration of 15 mM.
and 14.5 min for strains M-62 and M-64, respectively. This suggests that urea is capable of both entering and leaving yeast cells. To demonstrate the exit of urea directly, two parallel cultures of each strain (M-62 and M-64) were allowed to accumulate [14C]urea for 12 min. At this time the four cell samples were harvested by filtration, washed with prewarmed, preaerated medium, and resuspended in their original volume of fresh medium. At various times after resuspension samples were removed from the cultures and assayed either for the amount of radioactivity remaining within the cells (Fig. 4) or appearing in the medium. There is a reciprocal relationship between the gradual loss of urea from cells and its time-dependent accumulation in the medium (Fig. 4). Radioactive material observed in the cells and medium was demonstrated to be urea by its sensitivity to highly purified urease (Table 2). The data in Fig. 1 and 2 raise the possibility that production of a urea transport system is regulated in a fashion similar to that of the allantoin degradative system (2). The analogy between these two systems extends to their mutual repression when cultures are grown in the presence of a readily utilized amino acid (Fig. 5). Urea accumulation is greatest in cultures grown on proline as sole nitrogen source; they progressively lose this ability when grown on ammonia, aspartate, serine, or asparagine, respectively. It should also be noted (Fig. 5) that production of the transport system in a urea carboxylase-minus strain (M-62) is more
44
8
12
1
4
8
12
16
0242
20
24
28
23
32
36
MINUTES
FIG. 4. Loss of previously accumulated urea into the medium. Induced cultures (two from each strain) of M-64 were incubated for 12 mm in strainsM-6 the presence of 0.36 mM urea. At this time, 10 ml of each culture were harvested, washed with approximately 100 ml of prewarmed pre-aerated medium, and resuspended in their original volume of fresh medium. At the indicated times a sample of the cells (a) or medium (0) were obtained for each strain. It should be emphasized that cells were received from one culture and medium from an identical culture. A portion of the medium was counted directly in Aquasol. The cells were washed as described in Materials and Methods before being placed in Aquasol. The data are expressed as nanomoles of [14CJurea per milliliter of cells (original culture) or medium.
sensitive to the repressive effects of aspartate than is an allophanate hydrolase-minus strain (M-64). The reason behind this difference, however, is not presently known. The distinguishing characteristic of active transport against a concentration gradient as opposed to passive or facilitated diffusion is the energy requirement of the former process. To decide whether urea accumulation was the result of active transport or one of the two types of diffusion, the effects of various energy metabolism inhibitors upon this process were ascertained. Urea accumulation could be eliminated by arsenate, dinitrophenol, cyanide, and fluoride, clearly attesting the energy requirement of urea transport (Fig. 6). Not only is energy required to accumulate urea, but it is
J. BACTERIOL.
COOPER AND SUMRADA
574
TABLE 2. Urea accumulation and loss from Saccharomyces
cerevisiaea 103 counts/min per ml of culture
Source assayed
M-62
M-64
39.5 23.7
93.9 86.1
38.8
92.2
22.9
79.3
Urea incorporated into cells Urea lost from cells into the medium Urease-sensitive material in the cells Urease-sensitive material in the medium
aSamples of cells and medium similar to the 40-min points of Fig. 3 were treated with 1 mg of highly purified urease for 30 min. CO2 released upon acidification was then collected and its radioactivity content determined. SF-ILAb-?
A
M-62
PROLINE
4.0 3.2
2
~~~~AMMONIA
L-i2.4
_
I-~~~~~~~~~~~~~~~~~~~~~~~~~ W
10
12
16
14
-j 5.0
w
0
4.0
U, W
0
8
FIG. 6. Sensitivity of urea uptake to inhibitors of energy metabolism. This experiment was performed as described in Fig. 1 or 5. However, 2 min before beginning the assay an inhibitor was added. Inhibitors were used at the following final concentrations: arsenate, 5 mM; dinitrophenol, I mM; cyanide, 1 mM; and fluoride, 5 mM.
6.8 _AS PARTATE 0
6
MINUTES
0
a. z
4
2
4
6
8
10
12
14
16
SPA RTAT E ~~~~~~~~~~~~~A
2
WL cE 3.0 0
,*2.0
0
MINUTES
FIG. 5. Sensitivity of urea uptake to nitrogen repression. These experiments were performed in a manner similar to that used for the induced culture in Fig. 1A and the uninduced culture in Fig. lB except that in place of ammonia the nitrogen source used was that indicated at a concentration of 0.1%. The radioactive urea concentration was 0.36 mM.
also required to retain urea within the cell. This was shown (Fig. 7) by permitting a culture to accumulate urea for 16 min. At that time one-half of the culture was transferred to a second incubation vessel containing dinitrophe-
6
12
18
24 30 MINUTES
36
42
48
FIG. 7. Requirement of energy to retain accumulated urea within the cell. This experiment was performed as described for the induced culture of Fig. JA except that dinitrophenol (1 mM final concentration) was added at 16.5 min.
nol (1 mM final concentration). As depicted in this figure addition of dinitrophenol resulted in the immediate exit of urea that had been
previously accumulated. A Lineweaver-Burk plot of a urea concentration curve was used to determine the Km value
VOL. 121, 1975
V
UREA TRANSPORT
oo4/
V0.04 004
002
_
4
8
12
16
20
24
28
32
36
S
FIG. 8. Lineweaver-Burk plot of a urea contcentration curve. An induced culture of strain M-62 was used at a cell density of 45 Klett units. At zero time a 2.0-ml portion of the culture was added to an appropriate amount of radioactive urea (0.025 to 5.0 mM at a specific activity of 6.2 uCi/pmol). Incubation was carried out for 4 min and 1.0-ml samples were transferred to a membrane filter (Millipore Corp.) and washed with 32 ml of cold medium containing 20 mM urea. The K. value of 14 uM is the average of three experiments (12, 16, and 15 MM).
of the
urea transport system. This plot is biphasic, consisting of one linear portion yielding an apparent Km value of 14,uM and a second linear portion which begins at approximately 0.5 mM. At concentrations in excess of 0.5 mM there is a much greater rate of entry than expected. This likely results from the existence of two systems participating in urea uptake: an energy-dependent, low Km active transport system and an energy-independent, passive or facilitated diffusion system. Table 3 depicts a limited preliminary characterization of the urea uptake process occurring at an external concentration of 10 mM. At this concentration urea uptake is rapid, independent of induction by oxaluric acid, and insensitive to both repression and energy metabolism inhibitors.
DISCUSSION These data provide evidence for two pathways of urea uptake in S. cerevisiae. The first mode of uptake exhibits a Km value of 14 MM. Like the active transport systems of Escherichia coli, urea transport at low concentration is absolutely energy dependent (5) as is urea retainment within the cell (4). Production of the urea-active transport system appears to be subject to the same control exerted on the other enzymes of allantoin and urea metabolism; it is induced by allophanate and subject to repression by readily utilized amino acids. A second mode of uptake becomes apparent at external
575
urea concentrations above 0.5 mM. This uptake system acts rapidly reaching equilibrium in under 2 min. It does not require energy or pregrowth in the presence of inducer and is not sensitive to nitrogen repression. However, a note of caution is necessary because the accuracy and precision of the data in Table 3 are considerably lower than those reported in Fig. 1 to 8. The major problem, resulting in scatter of the data, is that urea taken up into the cells can rapidly diffuse out again during the wash procedure. This problem has been alleviated to an extent by the inclusion of urea (20 mM) in the washing medium, but until this problem is completely resolved the data in Table 3 must be considered as preliminary. The demonstration of 'urea rapidly entering the cell when present at high (10 mM) external concentrations may offer an explanation for differences observed in the induction kinetics of allophanate hydrolase and a-glucosidase. Lawther and Cooper (7) reported that 3 min elapse between addition of 10 mM urea and appearance of allophanate hydrolase activity. On the other hand, Kuo et al. (6) observe a lag of 10 to 20 min between addition of the inducer, maltose, and appearance of a-glucosidase activity. If maltose cannot enter the cell by facilitated or passive diffusion then a significant portion of the 10- to 20-min lag observed by the TABLE 3. Characteristics of urea uptake at 10 mM external concentrationa
['4Cjurea uptake (nmol) sample (min) per milliliter of culture.
.Time of Growth conditin
Uninduced Uninduced Induced Induced on asparagine Uninduced on asparagine Induced and DNP added before assay
2 14 14 14 14 14
16.2 12.3 13.2 17.8 17.2 13.5
aThe values reported here are from the 14-min point of experiments similar to those depicted in Fig. 1, 5, and 6. A single value is reported instead of the entire curve in the interest of brevity. However, it should be emphasized that the data are not as precise as those depicted in the above figures; the error is likely ± 10%. In these experiments the concentration of urea in the assay mixture was 10 mM at a specific activity of 1.2 ;Ci/umol. Also the harvested cells were washed with 8 ml of minimal medium containing 20 mM urea instead of 32 ml of 10 mM urea medium. This alteratiorn in washing procedure significantly improved the quality of the observed data but it is still not totally satisfactory.
576
COOPER AND SUMRADA
latter workers may elapse before the internal cellular concentration of maltose reaches the threshold value required to bring about induction. Consistent with this is the fact that maltose is transported by a specific inducible uptake system (8). The inducibility and sensitivity to nitrogen repression of the urea active transport system in S. cerevisiae should prove valuable in studies addressing the sequence of events occurring between synthesis of the presumed transport protein and its incorporation into the membrane of the cell. It is likely that these two events can be temporally separated using the methods previously developed to study allophanate hydrolase induction (Lawther and Cooper, J. Bacteriol., in press). If this can be done, an important tool will become available which in concert with genetic approaches may be brought to bear on this problem.
3. 4.
5.
6.
7.
8.
9.
ACKNOWLEDGMENTS 10.
We express gratitude to Ronald Kaback and Richard Abrams for their helpful comments. This work was supported by Public Health Research grants GM-19386 and GM-20693 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bossinger, J., R. P. Lawther, and T. G. Cooper. 1974. Nitrogen repression of the allantoin degradative enzymes in Saccharomyces cerevisiae. J. Bacteriol. 118:821-829. 2. Cooper, T. G., and R. P. Lawther. 1973. Induction of the allantoin degradative enzymes in Saccharormyces cere-
11.
12.
13.
J. BACTERIOL.
visiae by the last intermediate of the pathway. Proc. Nat. Acad. Sci. U.S.A. 70:2340-2344. Domnas, A. 1962. Amide metabolism in yeasts. The uptake of amide and amide-like compounds by yeast. J. Biochem. 52:149-154. Fields, K. L., and S. E. Luria. 1969. Effects of colicins El and K on transport systems. J. Bacteriol. 97:57-63. Kennedy, E. P. and G. A. Scarborough. 1967. Mechanism of hydrolysis of o-nitropheny ,8-galactoside in Staphylococcus aureus and its significance for theories of sugar transport. Proc. Nat. Acad. Sci. U.S.A. 58:225-228. Kuo, S. C., F. R. Cano, and J. 0. Lampen. 1973. Lomofungin, an inhibitor of ribonucleic acid synthesis in yeast protoplasts: its effect on enzyme formation. Antimicrob. Agents Chemother. 3:716-722. Lawther, R. P., and T. G. Cooper. 1973. Effects of inducer addition and removal upon the level of allophanate hydrolase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 55:1100-1104. Sols, A., C. Gancedo, and G. Delafuente. 1971. Energyyielding metabolism in yeasts, p. 290. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., New York. Sumrada, R., and T. G. Cooper, 1974. Oxaluric acid: a non-metabolizable inducer of the allantoin degradative enzymes in Saccharomyces cerevisiae. J. Bacteriol. 117:1240-1247. Whitney, P. A., and T. G. Cooper. 1972. Urea carboxylase and allophanate hydrolase: two components of a multienzyme complex in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 49:45-51. Whitney, P. A., and T. G. Cooper. 1972. Urea carboxylase and allophanate hydrolase: two components of ATP: urea amido-lyase in Saccharomyces cerevisiae. J. Biol. Chem. 247:1349-1353. Whitney, P. A., and T. G. Cooper. 1970. Requirement for HCO, by ATP:urea amido-lyase in yeast. Biochem. Biophys. Res. Commun. 40:814-819. Whitney, P. A., T. G. Cooper, and B. Magasanik. 1973. The induction of urea carboxylase and allophanate hydrolase in Saccharomyces cerevisiae. J. Biol. Chem. 248:6203-6209.
JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 571-576 Copyright i 1975 American Society for Microbiology
Urea Transport in Saccharomyces cerevisiae TERRANCE G. COOPER* AND ROBERTA SUMRADA Department of Biochemistry-FAS, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 11 October 1974
Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 ,M, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion. Saccharomyces cerevisiae can utilize urea as sole nitrogen source by degrading it in two steps to ammonia and CO2 (10). The enzymes responsible for this degradation are urea carboxylase and allophanate hydrolase (11). Their production is contingent upon the presence of allophanic acid (2, 13) or the gratuitous inducer oxaluric acid (9) and is subject to nitrogen repression when cells are grown on readily utilized amino acids (1). Although there is reasonable understanding of the biochemistry, genetics, and physiology of the urea-degradative enzymes, there is no information concerning how urea is taken into the cell. Domnas reported (3) that during growth on allantoin the urea generated appears in the medium suggesting its exit from the cell, but no further data are given. In view of this deficiency we have investigated the mechanisms by which urea is accumulated and report that two modes of uptake are operative. MATERIALS AND METHODS Strains and media. Two strains defective in urea metabolism were used in this work. M-62 lacks urea carboxylase and M-64 lacks allophanate hydrolase activity (11). These strains were used in place of the
gm pore size) (Millipore Corp.) and unless indicated otherwise washed four times with 8 ml of cold medium containing 10 mM urea. Washed filters were placed in 5 ml of Aquasol (New England Nuclear Corp.) scintillation fluid and counted 16 to 24 h later. The incubation time in Aquasol was needed to allow the filters to become transparent. Failure to do this results in unevenly quenched samples and loss of assay precision. The report by Leder and Perry (Fed. Proc. 26:394) that washing with cold medium leads to a rapid loss of accumulated substrates prompted evaluation of our washing procedures. The losses reported by these investigators do not occur in Saccharomyces treated with cold medium (Table 1). Note also that only 8 ml of medium are required to completely remove remaining external urea; 32 ml was routinely used, however, as a precaution. Unless otherwise indicated, all data are expressed as amounts of radioactive urea observed per milliliter of the original culture.
RESULTS A urea carboxylase-minus strain of S. cerevisiae (M-62) cannot accumulate ["C lurea when grown on minimal ammonia medium (closed circles) (Fig. 1A). However, if oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, is present during growth this strain is capable of urea accumulation. A quite different behavior is observed when this, experiment is repeated using an allophanate hydrolase-minus strain (M-64). [TC ]urea is accumulated in this strain even in the absence of exogenous gratuitous inducer (Fig. 1B). To decide whether oxaluric acid is functioning here as a urea or allophanate analogue the experiment in Fig. 1A was repeated using arginine as sole nitrogen source in place of ammonia. This assures a high intracellular concentration of urea during cell growth. The presence of large quantities of urea within cells did not significantly increase their ability to accumulate urea from the medium.
wild-type strain because urea taken into wild-type cells is rapidly metabolized to CO2 which is then released into the medium (12). The medium used here was that described earlier (2). Except where indicated otherwise the nitrogen source was 0.1% ammonium sulfate. Urea uptake assay. For assay of urea uptake cultures were grown at 30 C to a cell density of 1.3 x 107 cells per ml (45 Klett units). At zero time an 8.5-ml portion of the culture was transferred to a flask containing ['IC lurea (specific activity, 4.7 ;Ci/4mol). Incubation was carried out at 30 C in a shaking water bath under conditions identical to those used during growth. At appropriate times 1.0-ml samples were transferred to nitrocellulose membrane filters (0.45 71
TABLE 1. Washing procedures for assay of urea uptake Temp (C)
Wash solutiona
Minimal medium Minimal medium + 10 mM urea Minimal medium
Minimal medium (8 ml) Minimal medium (16 ml) Minimal medium (24 ml) Minimal medium (34 ml) a Volume of wash solution wise indicated.
36 A
4 4
22 4 4 4 4 was
nmol of urea/ml of culture 2 min
16 min
0.77 0.74
3.21 3.34
0.79
3.21
4 min
16 min
0.65 0.61 0.57 0.61
1.28 1.26 1.21 1.22
32 ml unless other-
levels of urea accumulated in cells grown on ammonia and arginine likely results from isotope dilution which arises from catabolism of arginine to urea. These data closely parallel those observed in studies demonstrating the contingency of allantoin degradative enzyme production upon the presence of allophanic acid. If indeed these data reflect an ability to transport urea into cells it should be possible to "chase" radioactive urea out of the cells by addition of a large excess of nonradioactive urea to the culture. This was observed for both strains (Fig. 3). Addition of excess nonradioactive urea to cultures actively accumulating [4C ]urea resulted in abrupt loss of radioactivity from the cells. The half-life of this loss was 9.0
M-62 o
/
3.0 -+OXLU° 2.4
O
2.4
z Cn
2.0
ai. C,)
o,1.8
1_j
-j
-j
1.2_
AMMONIA + OXLU
2.8
w
/
--OXLU *
uj
J. BACTERIOL.
COOPER AND SUMRADA
572
w
0(.
w
1.6
04 /
ARGININE
+ OXLU
0
1.2
-C06
w
2 B
a a.
Z
85
0
4
8 6 M-64
10
12
14 C)
w 0 2C
0.8
0.4
AGNNE ALONE
w
2
4
6
8
10
12
MINUTES FIG. 2. Effect of intracellular urea upon production of the urea uptake system. Three cultures of strain M-62 were grown respectively on minimal ammonia medium plus oxaluric acid (0), minimal arginine medium plus oxaluric acid (a) and minimal arginine medium alone (U). At a cell density of 45 Klett units each culture was harvested by filtration and resuspended in fresh, prewarmed, pre-aerated, minimal ammonia medium. Those cultures previously grown 8 4 6 10 12 14 16 2 in the presence of oxaluric acid also received oxaluric MINUTES acid after resuspension in fresh medium. The three FIG. 1. Uptake of urea in urea carboxylase, M-62 cultures were incubated in this medium for 30 min to (A) and allophanate hydrolase, M-64 (B) minus permit urea within the cells to leave. The time strains of Saccharomyces. Cultures of each strain required for this to occur was determined from the were grown on ammonia minimal medium in the data shown in Fig. 4. At the end of this incubation presence (0) or absence (-) of 0.5 mM oxaluric acid. period (zero time in the figure) urea uptake was Urea uptake assays were performed as indicated in assayed as described in Materials and Methods. Materials and Methods. External radioactive urea Failure to remove urea from cells grown on arginine concentration was 0.36 mM. using these procedures greatly decreased their ability to accumulate added radioactive urea. This is due to a However, this (Fig. 2) ability was observed if steady state amount already being present within the oxaluric acid was present during growth. It cells at the time of radioactive urea addition to the should be noted that the difference between the medium. -J
0
4
2
0 ~~~~~~M-64
IL 60 O
0 ~~~~~~M-62
3.2
0 50
a _j
573
UREA TRANSPORT
VOL. 121, 1975
D a
r EXCESS
UREA _j40 40- /ADDED
w 2
w
.5 30 U,
w
2.4
1.6
0.8
20
8
16
24
32
40
48
56
64
MINUTES FIG. 3. Reversibility of urea uptake in Saccharomyces. The conditions of this experiment are identical to those used for the induced cultures of Fig. 1A. However, at 16.5 min nonradioactive urea was added to the assay mixture to a final concentration of 15 mM.
and 14.5 min for strains M-62 and M-64, respectively. This suggests that urea is capable of both entering and leaving yeast cells. To demonstrate the exit of urea directly, two parallel cultures of each strain (M-62 and M-64) were allowed to accumulate [14C]urea for 12 min. At this time the four cell samples were harvested by filtration, washed with prewarmed, preaerated medium, and resuspended in their original volume of fresh medium. At various times after resuspension samples were removed from the cultures and assayed either for the amount of radioactivity remaining within the cells (Fig. 4) or appearing in the medium. There is a reciprocal relationship between the gradual loss of urea from cells and its time-dependent accumulation in the medium (Fig. 4). Radioactive material observed in the cells and medium was demonstrated to be urea by its sensitivity to highly purified urease (Table 2). The data in Fig. 1 and 2 raise the possibility that production of a urea transport system is regulated in a fashion similar to that of the allantoin degradative system (2). The analogy between these two systems extends to their mutual repression when cultures are grown in the presence of a readily utilized amino acid (Fig. 5). Urea accumulation is greatest in cultures grown on proline as sole nitrogen source; they progressively lose this ability when grown on ammonia, aspartate, serine, or asparagine, respectively. It should also be noted (Fig. 5) that production of the transport system in a urea carboxylase-minus strain (M-62) is more
44
8
12
1
4
8
12
16
0242
20
24
28
23
32
36
MINUTES
FIG. 4. Loss of previously accumulated urea into the medium. Induced cultures (two from each strain) of M-64 were incubated for 12 mm in strainsM-6 the presence of 0.36 mM urea. At this time, 10 ml of each culture were harvested, washed with approximately 100 ml of prewarmed pre-aerated medium, and resuspended in their original volume of fresh medium. At the indicated times a sample of the cells (a) or medium (0) were obtained for each strain. It should be emphasized that cells were received from one culture and medium from an identical culture. A portion of the medium was counted directly in Aquasol. The cells were washed as described in Materials and Methods before being placed in Aquasol. The data are expressed as nanomoles of [14CJurea per milliliter of cells (original culture) or medium.
sensitive to the repressive effects of aspartate than is an allophanate hydrolase-minus strain (M-64). The reason behind this difference, however, is not presently known. The distinguishing characteristic of active transport against a concentration gradient as opposed to passive or facilitated diffusion is the energy requirement of the former process. To decide whether urea accumulation was the result of active transport or one of the two types of diffusion, the effects of various energy metabolism inhibitors upon this process were ascertained. Urea accumulation could be eliminated by arsenate, dinitrophenol, cyanide, and fluoride, clearly attesting the energy requirement of urea transport (Fig. 6). Not only is energy required to accumulate urea, but it is
J. BACTERIOL.
COOPER AND SUMRADA
574
TABLE 2. Urea accumulation and loss from Saccharomyces
cerevisiaea 103 counts/min per ml of culture
Source assayed
M-62
M-64
39.5 23.7
93.9 86.1
38.8
92.2
22.9
79.3
Urea incorporated into cells Urea lost from cells into the medium Urease-sensitive material in the cells Urease-sensitive material in the medium
aSamples of cells and medium similar to the 40-min points of Fig. 3 were treated with 1 mg of highly purified urease for 30 min. CO2 released upon acidification was then collected and its radioactivity content determined. SF-ILAb-?
A
M-62
PROLINE
4.0 3.2
2
~~~~AMMONIA
L-i2.4
_
I-~~~~~~~~~~~~~~~~~~~~~~~~~ W
10
12
16
14
-j 5.0
w
0
4.0
U, W
0
8
FIG. 6. Sensitivity of urea uptake to inhibitors of energy metabolism. This experiment was performed as described in Fig. 1 or 5. However, 2 min before beginning the assay an inhibitor was added. Inhibitors were used at the following final concentrations: arsenate, 5 mM; dinitrophenol, I mM; cyanide, 1 mM; and fluoride, 5 mM.
6.8 _AS PARTATE 0
6
MINUTES
0
a. z
4
2
4
6
8
10
12
14
16
SPA RTAT E ~~~~~~~~~~~~~A
2
WL cE 3.0 0
,*2.0
0
MINUTES
FIG. 5. Sensitivity of urea uptake to nitrogen repression. These experiments were performed in a manner similar to that used for the induced culture in Fig. 1A and the uninduced culture in Fig. lB except that in place of ammonia the nitrogen source used was that indicated at a concentration of 0.1%. The radioactive urea concentration was 0.36 mM.
also required to retain urea within the cell. This was shown (Fig. 7) by permitting a culture to accumulate urea for 16 min. At that time one-half of the culture was transferred to a second incubation vessel containing dinitrophe-
6
12
18
24 30 MINUTES
36
42
48
FIG. 7. Requirement of energy to retain accumulated urea within the cell. This experiment was performed as described for the induced culture of Fig. JA except that dinitrophenol (1 mM final concentration) was added at 16.5 min.
nol (1 mM final concentration). As depicted in this figure addition of dinitrophenol resulted in the immediate exit of urea that had been
previously accumulated. A Lineweaver-Burk plot of a urea concentration curve was used to determine the Km value
VOL. 121, 1975
V
UREA TRANSPORT
oo4/
V0.04 004
002
_
4
8
12
16
20
24
28
32
36
S
FIG. 8. Lineweaver-Burk plot of a urea contcentration curve. An induced culture of strain M-62 was used at a cell density of 45 Klett units. At zero time a 2.0-ml portion of the culture was added to an appropriate amount of radioactive urea (0.025 to 5.0 mM at a specific activity of 6.2 uCi/pmol). Incubation was carried out for 4 min and 1.0-ml samples were transferred to a membrane filter (Millipore Corp.) and washed with 32 ml of cold medium containing 20 mM urea. The K. value of 14 uM is the average of three experiments (12, 16, and 15 MM).
of the
urea transport system. This plot is biphasic, consisting of one linear portion yielding an apparent Km value of 14,uM and a second linear portion which begins at approximately 0.5 mM. At concentrations in excess of 0.5 mM there is a much greater rate of entry than expected. This likely results from the existence of two systems participating in urea uptake: an energy-dependent, low Km active transport system and an energy-independent, passive or facilitated diffusion system. Table 3 depicts a limited preliminary characterization of the urea uptake process occurring at an external concentration of 10 mM. At this concentration urea uptake is rapid, independent of induction by oxaluric acid, and insensitive to both repression and energy metabolism inhibitors.
DISCUSSION These data provide evidence for two pathways of urea uptake in S. cerevisiae. The first mode of uptake exhibits a Km value of 14 MM. Like the active transport systems of Escherichia coli, urea transport at low concentration is absolutely energy dependent (5) as is urea retainment within the cell (4). Production of the urea-active transport system appears to be subject to the same control exerted on the other enzymes of allantoin and urea metabolism; it is induced by allophanate and subject to repression by readily utilized amino acids. A second mode of uptake becomes apparent at external
575
urea concentrations above 0.5 mM. This uptake system acts rapidly reaching equilibrium in under 2 min. It does not require energy or pregrowth in the presence of inducer and is not sensitive to nitrogen repression. However, a note of caution is necessary because the accuracy and precision of the data in Table 3 are considerably lower than those reported in Fig. 1 to 8. The major problem, resulting in scatter of the data, is that urea taken up into the cells can rapidly diffuse out again during the wash procedure. This problem has been alleviated to an extent by the inclusion of urea (20 mM) in the washing medium, but until this problem is completely resolved the data in Table 3 must be considered as preliminary. The demonstration of 'urea rapidly entering the cell when present at high (10 mM) external concentrations may offer an explanation for differences observed in the induction kinetics of allophanate hydrolase and a-glucosidase. Lawther and Cooper (7) reported that 3 min elapse between addition of 10 mM urea and appearance of allophanate hydrolase activity. On the other hand, Kuo et al. (6) observe a lag of 10 to 20 min between addition of the inducer, maltose, and appearance of a-glucosidase activity. If maltose cannot enter the cell by facilitated or passive diffusion then a significant portion of the 10- to 20-min lag observed by the TABLE 3. Characteristics of urea uptake at 10 mM external concentrationa
['4Cjurea uptake (nmol) sample (min) per milliliter of culture.
.Time of Growth conditin
Uninduced Uninduced Induced Induced on asparagine Uninduced on asparagine Induced and DNP added before assay
2 14 14 14 14 14
16.2 12.3 13.2 17.8 17.2 13.5
aThe values reported here are from the 14-min point of experiments similar to those depicted in Fig. 1, 5, and 6. A single value is reported instead of the entire curve in the interest of brevity. However, it should be emphasized that the data are not as precise as those depicted in the above figures; the error is likely ± 10%. In these experiments the concentration of urea in the assay mixture was 10 mM at a specific activity of 1.2 ;Ci/umol. Also the harvested cells were washed with 8 ml of minimal medium containing 20 mM urea instead of 32 ml of 10 mM urea medium. This alteratiorn in washing procedure significantly improved the quality of the observed data but it is still not totally satisfactory.
576
COOPER AND SUMRADA
latter workers may elapse before the internal cellular concentration of maltose reaches the threshold value required to bring about induction. Consistent with this is the fact that maltose is transported by a specific inducible uptake system (8). The inducibility and sensitivity to nitrogen repression of the urea active transport system in S. cerevisiae should prove valuable in studies addressing the sequence of events occurring between synthesis of the presumed transport protein and its incorporation into the membrane of the cell. It is likely that these two events can be temporally separated using the methods previously developed to study allophanate hydrolase induction (Lawther and Cooper, J. Bacteriol., in press). If this can be done, an important tool will become available which in concert with genetic approaches may be brought to bear on this problem.
3. 4.
5.
6.
7.
8.
9.
ACKNOWLEDGMENTS 10.
We express gratitude to Ronald Kaback and Richard Abrams for their helpful comments. This work was supported by Public Health Research grants GM-19386 and GM-20693 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bossinger, J., R. P. Lawther, and T. G. Cooper. 1974. Nitrogen repression of the allantoin degradative enzymes in Saccharomyces cerevisiae. J. Bacteriol. 118:821-829. 2. Cooper, T. G., and R. P. Lawther. 1973. Induction of the allantoin degradative enzymes in Saccharormyces cere-
11.
12.
13.
J. BACTERIOL.
visiae by the last intermediate of the pathway. Proc. Nat. Acad. Sci. U.S.A. 70:2340-2344. Domnas, A. 1962. Amide metabolism in yeasts. The uptake of amide and amide-like compounds by yeast. J. Biochem. 52:149-154. Fields, K. L., and S. E. Luria. 1969. Effects of colicins El and K on transport systems. J. Bacteriol. 97:57-63. Kennedy, E. P. and G. A. Scarborough. 1967. Mechanism of hydrolysis of o-nitropheny ,8-galactoside in Staphylococcus aureus and its significance for theories of sugar transport. Proc. Nat. Acad. Sci. U.S.A. 58:225-228. Kuo, S. C., F. R. Cano, and J. 0. Lampen. 1973. Lomofungin, an inhibitor of ribonucleic acid synthesis in yeast protoplasts: its effect on enzyme formation. Antimicrob. Agents Chemother. 3:716-722. Lawther, R. P., and T. G. Cooper. 1973. Effects of inducer addition and removal upon the level of allophanate hydrolase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 55:1100-1104. Sols, A., C. Gancedo, and G. Delafuente. 1971. Energyyielding metabolism in yeasts, p. 290. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., New York. Sumrada, R., and T. G. Cooper, 1974. Oxaluric acid: a non-metabolizable inducer of the allantoin degradative enzymes in Saccharomyces cerevisiae. J. Bacteriol. 117:1240-1247. Whitney, P. A., and T. G. Cooper. 1972. Urea carboxylase and allophanate hydrolase: two components of a multienzyme complex in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 49:45-51. Whitney, P. A., and T. G. Cooper. 1972. Urea carboxylase and allophanate hydrolase: two components of ATP: urea amido-lyase in Saccharomyces cerevisiae. J. Biol. Chem. 247:1349-1353. Whitney, P. A., and T. G. Cooper. 1970. Requirement for HCO, by ATP:urea amido-lyase in yeast. Biochem. Biophys. Res. Commun. 40:814-819. Whitney, P. A., T. G. Cooper, and B. Magasanik. 1973. The induction of urea carboxylase and allophanate hydrolase in Saccharomyces cerevisiae. J. Biol. Chem. 248:6203-6209.
