YEAST
VOL.7: 933-94 1 (1 99 1)
A High-affinity Uptake System for Branched-chain Amino Acids in Saccharomyces cerevisiae S 0 R E N TULLIN, CLAES GJERMANSEN A N D MORTEN C. KIELLAND-BRANDT* Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received 29 January 1991;accepted 18 June 1991
In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched-chain amino acid uptake. Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics. This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected. Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, Lisoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids. We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild-type allele is denoted BAPI for branched-chain amino acid permease. The BAPI-dependent system is different from the general amino acid permease. KEY WORDS - BAPI; branched-chain amino acids; transport; permease; uptake; L-isoleucine; L-leucine; L-valine; sulfometuron methyl; yeast.
INTRODUCTION Amino acid transport into yeast cells is an active and essentially unidirectional process driven by the electrochemical proton gradient (Seaston et al., 1973; Horak, 1986). The well-characterized general amino acid permease (GAP) is the only general uptake system identified so far in Saccharomyces cerevisiae, whereas a number of specific carriers have been found each responsible for transport of a small group of related amino acids (reviews: Horak, 1986; Cartwright et al., 1989). The high-affinity transport system for L-arginine is an example of a carrier transporting a group of related amino acids, as all three normal basic amino acids and L-canavanine are transported by this system (Grenson etal., 1966; Grenson, 1966). Concerning synthesis and inactivation of carriers, the GAP and the high-affinity L-proline carrier, encoded by the PUT4 gene, are subject to nitrogen*Addressee for correspondence 0749-503X/9 1/090933-O9$05.00 0 1991 by John Wiley & Sons Ltd
catabolite regulation (Cartwright et al., 1989). General amino acid control of carriers has to our knowledge not been published. However, the sequence published for the promoter of the HIP1 gene encoding the histidine permease (Tanaka and Fink, 1985) contains two TGACTC elements. In some cases it has been possible to distinguish kinetically two uptake components. L-leucine uptake, for example, can be explained by two parallel systems, both obeying Michaelis-Menten kinetics (Ramos et d., 1980; Kotliar and Ramos, 1983; Wainer et al., 1988): a high-affinity, low-capacity system and a low-affinity system with high capacity. Studies on amino acid transport in Schizosaccharomyces pombe revealed that L-phenylalanine, Lisoleucine, L-valine and L-cysteine are competitive inhibitors of L-leucine uptake (Sychrova et al., 1989), suggesting that these amino acids are substrates for a L-leucine transporting carrier. The present study describes the identification of a system responsible for high-affinity uptake of Lisoleucine. L-leucine and L-valine.
934 MATERIALS AND METHODS Strains
The parental strain for the mutagenesis was S288C ( M A T a SUC2 ma1 gal2 CUPI). Strain X2180-1A (MATa SUC2 malga12 CUP1) was used as parent in tetrad analysis. Other strains were derived as described. Media
Synthetic minimal medium MA (minimal ammonia) was made by adding 10 g succinic acid, 6 g NaOH, 5 g (NH,)*SO, and 1.7 g Bacto Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco) to 1 litre of H,O. MP is MAmedium containing 2g/l L-proline instead of the (NH,),SO,, Synthetic complete medium, SC, was MA medium supplemented with various nutrients: 20 mg/l adenine sulfate, 20 mg/l L-arginine hydrochloride, 300 mg/l L-aspartic acid, 100 mg/l L-glutamic acid, 20 mg/l L-histidine hydrochloride, 30 mg/l Lisoleucine, 30 mg/l L-leucine, 30 mg/l L-lysine hydrochloride, 60 mg/l L-methionine, 50 mg/l Lphenylalanine, 375 mg/l L-serine, 600 mg/l Lthreonine, 20 mg/l L-tryptophan, 30 mg/l L-tyrosine, 20 mg/l uracil and 30 mg/l L-valine. SC-Val-Ile-Leu was SC medium without L-valine,L-isoleucineand Lleucine. Complex medium with glucose was YPD YO Difco Bacto Yeast Extract, 2% Difco Bacto Peptone, 2% glucose). YPG contained 1% Difco Bacto Yeast Extract, 2% Difco Bacto Peptone, 3% glycerol. YPD+ SM was YPD medium containing Sulfometuron Methyl (N-[(4,6-dimethylpyrimidin2 - yl) - aminocarbonyl] - 2 - methoxycarbonylbenzenesulfonamide, 30 mg/l or 75 mg/l), kindly provided by S. C. Falco, E. I. Du Pont de Nemours & Company, Wilmington, Delaware). All solid media contained 2% Difco Bacto Agar. Mutagenesis and isolation of mutants sensitive to sulfometuron methyl on YPD
Cells of strain S288C were grown in liquid YPD to early stationary phase. Ultraviolet (UV) muta-
genesis was carried out either by plating the cells on YPG and irradiating them in situ or by irradiation of 2 x lo7cells, suspended in 20 ml water in a Petri dish, under agitation. In the latter case, enrichment for SM-sensitive cells was performed by heat killing of exponentially growing cells according to Walton et al. (1979): The cells were harvested, resuspended in 1 ml YPD and grown overnight in darkness to
S. TULLIN ET AL.
stationary phase. After 100-fold dilution in YPD + SM (30 mg/l), cells were grown for 5 h at 37°C (log phase) and heated to 50°C for 7 min. In pilot experiments in YPD, this treatment killed 99.6% of logarithmically growing cells, while 63% of stationary-phase cells survived. Cells were plated on YPG to give 200 colonies/plate. SM-sensitive mutants were identified by replica plating to YPD SM (30 mg/l).
+
Tetrad analysis Tetrad analysis was performed as described by Mortimer and Hawthorne (1966). Mutant and wild type were distinguished by replica plating onto YPD SM (75 mg/l) and reading of the plates after 1 day at 30°C.
+
Uptake assay In all experiments, exponentially growing cells were harvested and assayed for their uptake of a I4Clabelled amino acid. For most experiments an overnight culture in SC medium was diluted into the same medium and grown at 30°C for 5-6 h until an OD,, of 0.5 was attained. Uptake kinetics were assayed in SC-Ile-Leu-Val medium, while inhibition of branched-chain amino acid uptake by a non-labelled amino acid was ma y e d in MA medium. Exponentially growing cells were harvested by centrifugation, washed and resuspended in the medium indicated above. Two hundred microlitres of cells (- 2 mg dry wei ht) were added to 300 p1 medium containing the ',Clabelled amino acid and incubated at 25°C. After 45 min, the assay was stopped by the addition of 900 p1 of an ice-cold 15&250 mM solution of the same amino acid in unlabelled form. The cells were harvested after 0-5 min by centrifugation in a microfuge, washed three times with cold SC medium and counted. Uptake rates are expressed relatively to cell dry weight obtained by drying at 104°C for 18 h. The data presented are the average of three experiments; the average variation was less than 5%. In the experiments where uptake was assayed as a function of extracellular substrate concentration, 16 ~ different concentrations in the range of 2 5 p to 8 mM were used. All radioactive compounds were obtained from New England Nuclear: ~-[ureido-'~C]citrulline (NEC-214), ~-[~~C(U)]isoleucine (NEC-278E), L['4C(U)]leucine (NEC-279E) and ~-['~C(U)]valine (NEC-29 1E).
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
Analysis of the kinetic experiments was carried out using the Enzfitter program distributed by Elsevier BIOSOFT, 68 Hills Road, Cambridge, UK. Proportional weighing was used, i.e. the standard deviation of the determination of uptake rate
J=
935
was taken to be proportional to the uptake rate. Curves in the Eadie-Hofstee presentations (Jversus J / [ S ] ,where J is the uptake rate and [S] is the extracellular substrate concentration) were drawn by the Enzfitter program according to the equation:
J J -KTI--KT2-+JM,+JM2+ PI [SI 2
which was derived by adding two Michaelis-Menten equations and solving for J as a function of J / [ S ] .In the equation JMland KTIare capacity and affinity, respectively, for system 1 , and J M 2and KT2 are capacity and affinity, respectively, for system 2.
because only few generations of growth took place before plating.
Transport assay The uptake assay was designed for accuracy of sampling time. Transport of the labelled amino acid was stopped by the addition of an ice-cold solution Ammonia repression of the GAP of the same amino acid in unlabelled form. Control For the determination of ammonia repression of experiments with I4C-labelledL-valine, L-isoleucine the GAP (Garrett, 1989), an overnight culture in SC or L-leucine in strain S288C show that this approach medium was washed, diluted and transferred to MP efficiently blocks influx for at least 1 h (data not or MA medium. The cells were grown at 30°C for 5- shown). In addition, no detectable efflux can be 6 h until an OD,,, of 0.5 was attained. GAP activity seen. Washing of a large number of samples can was assayed by L-citrulline uptake (Grenson et al., therefore be carried out simultaneously. Branched1970) in fresh MA or MP medium. chain amino acid accumulation in strain S288C was tested for linearity in time. It was linear for at least 10 min (data not shown). RESULTS Uptake was assayed in defined media (SC, MA or Isolation of mutants with impaired uptake of the MP). The intention was to study uptake from a branched-chain amino acids medium of a composition close to that in which the Sulfometuron methyl (SM) impedes growth of S. cells had been growing. In the experiments where cerevisiue wild-type strains on medium lacking L- influx was assayed as a function of external subisoleucine and/or L-valine by inhibiting the aceto- strate concentration (Figures 1-3), SC-Val-Ile-Leu hydroxyacid synthase, encoded by the ILV2 gene was chosen as assay medium. Transport of the (Falco and Dumas, 1985). In contrast, SM has little branched-chain amino acids through carriers with effect when added to complete or complex medium, preference for amino acids other than L-valine, Lbecause most yeast strains can utilize branched- isoleucine and L-leucine should then be reduced to a chain amino acids present in the medium and are as minimum. A disadvantage is that the KT values prea consequence not dependent on de novo synthesis of sented in Table 1 are apparent values because of L-isoleucine and L-valine. In order to isolate possible competitive inhibition by other amino mutants with impaired uptake of either amino acid, acids. However, as will be shown later, the BAP1UV-induced mutants sensitive to SM on complex dependent system is virtually unaffected by the medium were isolated as described in Materials and presence of the additional amino acids. Methods. In the first experiment, cells were plated on YPG and UV-irradiated to 15% survival. By The BAPl gene replica plating of 10000 colonies to YPD+SM, Ten mutants, isolated as described above, were four mutants sensitive to SM were isolated, includ- assayed for their ability to accumulate brancheding C2901 to be described below. In another chain amino acids (data not shown). Three mutants, experiment, where thermal selection followed muta- including one called C2901, were identified with genesis, 14 SM-sensitive mutants were found among impaired uptake of all three branched-chain amino 4000 colonies. Even in the latter experiments the acids. None of the other seven mutants was affected mutants could be considered as being independent, in the uptake of any of the three amino acids, i.e.
936
S. TULLIN ET AL.
1 [VALINE] (mM)
0
200
400
600
800
2
3
4
5
6
[ISOLEUCINE] (mM)
1000
1200
J, / [VALINE] (nmolxgcelts*l. min-l* mM-')
J,l[ISOLEUCINE](nmol =9cell;1"min-1'mM-1)
Figure 2. (a) Influx of ~-isoleucine(J,)as a function of the external ~-isoleucineconcentration in strain S288C (+) and strain C2901(0). Assay medium was SC-Val-Ile-Leu.(b) EadieHofstee plot of the data given in (a) for strain S288C (+) and strain C2901 (0).
Figure 1. (a) Influx of ~-valine(J,)as a function of the external L-valine concentration in strain S288C (+) and strain C2901 (0). Assay medium was SC-Val-Ile-Leu.(b) Eadie-Hofsteeplot of the data given in (a) for strain S288C. (c) Eadie-Hofsteeplot of the data given in (a) for strain C2901.
we found no mutant defective for transport of only one or two branched-chain amino acids. For the primary genetic characterization, auxotrophic markers were avoided. Upon sporulation of the diploid X2180-1A x C2901, a 2:2 segregation of sensitivity to resistance to SM (75mg/l) on YPD was observed in 19 of 20 tested tetrads. Moreover, impaired uptake of branched-chain amino acids cosegregated with SM-sensitivity in four tested tetrads (data not shown). These findings indicate that a single Mendelian gene is affected in the mutant C2901. The wild-type gene is denoted BAPl
(branched-chain amino acid permease). The segregation of SM resistance turned out to be complicated in non-isogenic crosses (data not shown). Uptake of radioactively labelled isoleucine, valine and leucine in the diploids C3391 (BAPIIBAPZ), C3392 (BAPllbapl) and C3393 (bapllbapl) was compared. Table 1 shows that the mutant gene bapl is recessive. In contrast, bapl showed a semidominant phenotype on YPD+SM plates. It may be noted that the wild-type diploid C3391 has a lower uptake relative to cell mass than the wild-type haploids S288C and X2180-1A. Kinetics of L-valine,L-isoleucineand L-leucineuptake The kinetics were investigated in mutant strain C2901 and the parental strain S288C (Figures 1-3; Table 2). A straight line in an Eadie-Hofstee representation of the data is consistent with the presence of a single system obeying Michaelis-Menten kinetics. In the case of a curvature in the EadieHofstee representation of the experimental data, the kinetic parameters giving the best fitting curve for two systems in a J vs J/[S]plot were obtained using the Enzfitter program. In all those cases (Figures 2
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
o
i
z
i
4
i
k
i
i
[LEUCINE] (mM)
- , 0
I
100
200
J, /[LEUCINE]
0
100 J, I
-
400
300
500
(nmol=gce~l;'- min". m W ' )
200
[LEUCINE] (nmol.gcell;'
300
400
= min-', mM-')
Figure 3. (a) Influx of L-leucine (J,)as a function of the external L-leucine concentration in strain S288C (+) and strain C2901 (0). Assay medium was SC-Val-Ile-Leu. (b) Eadie-Hofstee plot ofthe data given in (a) for strain S288C. (c) Eadie-Hofstee plot of the data given in (a) for strain C2901.
and 3), the curves coincided with the experimental data, i.e. the observed kinetics could be explained by two parallel systems obeying Michaelis-Menten kinetics. Figures la, 2a and 3a show that accumulation of all three branched-chain amino acids is affected in the mutant, the effect being most pronounced for Lvaline. Eadie-Hofstee representation shows that Lvaline uptake in the mutant and the wild type fits a model of one system obeying Michaelis-Menten kinetics (Figures Ib and lc). The non-linear EadieHofstee representation of the L-isoleucine influx
937
data for the wild type and the close fitting curve (Figure 2b) obtained with the equation for two systems with the kinetic parameters presented in Table 2 suggest that L-isoleucine uptake is mediated by two systems obeying Michaelis-Menten kinetics in the wild type. In the mutant, on the other hand, Lisoleucine accumulation can be explained by one system obeying Michaelis-Menten kinetics. The system remaining in the mutant has the same kinetic parameters as the low-affinity system identified in the wild type (Figure 2b, Table 2). L-leucine accumulation is consistent with two systems, each obeying Michaelis-Menten kinetics, in both the mutant and the wild type. Free diffusion or a system with very high KT is responsible for the low-affinity component common to the wild type and the mutant (Figures 3b,c; Table 1). Since the KT values for this (these) low-affinity system(s) obtained from the computer anaylsis were larger than 1 M, the diffusion constant ( D = J M K Tis ) presented in Table 1, rather than the estimated KT and JMvalues. There are two obvious alternative interpretations of the data for L-valine accumulation. The carrier identified in the wild type is absent in the mutant, leaving a system which was too weak to be detected when superimposed with the stronger system, or the transport system in the mutant is a mutated version of the wild-type carrier with reduced activity. The kinetic parameters presented in Table 1 reveal a diffusion or very low-affinity component of Lleucine uptake, which is identical in the mutant and the wild type. The medium-affinity system in the mutant appears to be a mutated version of the highaffinity carrier found in the wild type. However, it cannot be excluded that it is a third system, masked in the wild type by the other systems. Inhibition of L-valine, L-isoleucine and L-leucine uptake by various amino acids Uptake of L-valine, L-isoleucine and L-leucine at 0.2 mM was assayed in the presence of various amino acids at 10 mM in strains S288C and C2901 (Table 3). The absolute increase in the uptake of each amino acid when the uptake from SC-Val-Ile-Leu (Figures 1-3) iscompared to that from SD (footnote of Table 3), is almost the same in the mutant and the wild type. This indicates that the amino acids in SCVal-Ile-Leu are not inhibiting uptake through the BAPI-dependent system and that the low-affinity carrier(s) for the branched-chain amino acids may be rather non-specific.
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Table 1. Uptake from SC medium of 14C-labelledL-valine, L-isoleucine and L-leucine (nmol gcells-l min-I). Average of two independent experiments.The average deviation was less than 5%
Genotype
Strain S228C X2 180-1A C290 1 C3003 c 3 137 C339 1 C3392 c3393
Source/reference
MATa M A Ta MATa bapl MATaIMA TaBAPllbapl MATa bapl MATaIMATa BAPIIBAPI MATaIMATa BAPljbapl MATaIMATa bupllbapl
Uptake of Val Ile Leu
Mortimer and Johnston (1986) Mortimer and Johnston (1986) Mutant of S288C, this study X2 180-1A x C2901 Spore clone of C3003
121 283 103 245 15 29 65 155 12 25 79 147 66 155 16 25
S288C x X2180-1A S288C x C3137 C2901 x C3137
550 500 88 373 47 387 347 56
Table 2. Apparent kinetic parameters of L-valine, L-isoleucine and L-leucine uptake in synthetic growth medium in strain S288C and strain C2901 calculated from the data presented in Figures 1-3. KT values are expressed in mM, JMvalues in nmol g-l min-l and the diffusion constant D in nmol g-l min-' mM-l Strain S288C(BAPI) C2901 (bapl) S288C (BAPI) C2901 (bapl) S288C (BAPI) C2901 (bapl)
Uptakeof Val Val Ile Ile Leu Leu
D
KTI
JMl
KTz
JMz
2.0 7.2
2400 950
-
-
-
-
-
-
0.3 1 4.2
540 2000
-
0.16 0.94
970 360
Uptake in the wild type in the presence of various amino acids (Table 3 ) reveals similar inhibition patterns for L-valine, L-isoleucine and L-leucine transport. L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine inhibit in all cases, to various extents. These findings suggest that highaffinity uptake of branched-chain amino acids in the wild type is carried out by one carrier or by several carriers with similar inhibition patterns. Moreover, L-methionine inhibits L-valine uptake to 38%, while L-glycine does not inhibit in any case. Assuming one high-affinity carrier with the kinetic parameters for L-valine, L-isoleucine and L-leucine uptake as presented in Table 2 (KT, and J M ,for strain S288C), Lisoleucine and L-leucine should be good competitors of L-valine transport. On the other hand, only weak inhibition of L-isoleucine transport and in particular L-leucine transport by L-valine is expected. These assumptions fit the data. However, the assumption causes difficulties in explaining why uptake of L[I4C]isoleucine in the presence of excess unlabelled
11
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-
-
-
-
-
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54 53
L-isoleucine at 10 mM is about ten-fold lower than the uptake in the presence of L-leucine at 10 mM and vice versa (Table 3). In strain C2901, the competitors investigated have weak and similar effects, suggesting that the system(s) responsible for branched-chain amino acid uptake in the mutant are relatively unspecific, low-affinity system(s).
G A P repression in the bapl mutant C2901 Recently, a new amino acid uptake mutant designated aatl was described (Garrett, 1989). When combined with a leu2 allele, aatl makes yeast unable to grow on rich medium or minimal medium supplemented with certain amino acids. Moreover, GAP activity was not repressed by ammonia in the mutant. It is an obvious possibility that aatl mutants are sensitive to SM on complex medium. To determine whether bapl has an aatl-like phenotype, ammonia
939
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
Table 3. Uptake of ''C-labelled L-valine, L-isoleucine or L-leucine at 0.2 mM in the presence of various non-radioactive amino acids at 10 mM. Numbers represent the rate of uptake as a per cent of the uptake in the absence of competitor
S288C ( B A P I ) Potential competitor None Ala Arg ASP CYS Glu GlY His Ile Leu LYS Met Phe Pro Ser Thr Trp Val Citt
Val 1oo*
4.8 90 119 20 74 104 87 7.0 5.8 83 38 141 86 94 76 36 7.0 ND
Uptake of Ile
loo* 18 100 112 54 98 89 98 3.8 30 112 103 96 87 89 80 64 47 ND
C2901 (bupl)
Leu
Val
Uptake of Ile
Leu
1 oo* 26 93 91 59 81 82 77 48 4.8 64 123 149 126 98 79 68 59 ND
loo* 21 ND ND 52 ND ND ND 21 20 ND 31 ND ND ND ND ND 34 102
loo* 37 ND ND 68 ND ND ND 39 52 ND 60 ND ND ND ND ND 42 90
1 oo* 21 ND ND 85 ND ND ND 26 12 ND 42 ND ND ND ND ND 58 113
*Absolute rates of uptake in this row, i.e. in the absence of competitor, were 300, 400,750,75,200 and 250nmol g - ' min-', respectively. tL-ci trulline. ND, not determined.
repression of the GAP in strains S288C and C2901 was investigated. Since L-citrulline is known to enter the cell solely through the GAP (Grenson et af., 1970), L-citrulline uptake was assayed after growth in MP and MA medium for 5 h. Table 4 shows that ammonia repression of GAP activity in the bapl mutant is identical to the repression seen in the wild type, suggesting that bapl is different from the aatl mutation.
DISCUSSION The present investigation defines a Mendelian gene, BAP1, by a mutation resulting in sensitivity to SM on the complex medium YPD. The bapl mutation causes a defect in high-affinity uptake of the branched-chain amino acids. The bapl mutation is distinct from the aatl mutation described by Garrett (1989).
Table 4. L-citrulline uptake (nmol g-' min-.') a t 0.1 mM in strains C2901 and S288C. Cells were grown in either MA or MP medium for 5 h and assayed for the uptake of L-citrulline Nitrogen source Ammonia Proline
S288C ( B A P 1 )
C2901 (bapl)
140 5200
160 4900
The kinetic parameters in Table 2 suggest that the low-affinity component for L-leucine uptake in the bapl mutant is the same as the low-affinity system in the wild type. Also, the kinetic parameters for the single uptake system for L-isoleucine in the mutant are similar to those for the low-affinity system in the wild type, suggesting that they are identical systems. This implies that the bapl mutation does not affect
940 low-affinity uptake of L-leucine and L-isoleucine.On the basis of this interpretation, the impaired uptake of branched-chain amino acids in the bapl mutant can be explained in two ways, with overlapping implications. One possibility is that a mutant version of a bapldependent system is responsible for L-valine uptake in the mutant and for medium-affinity L-leucine uptake in the mutant. A corresponding change in the system for high-affinity L-isoleucine uptake might well reduce transport to a level where identification would be difficult as a consequence of superposition with the low-affinity system. Altogether, this hypothesis implies that one permease is responsible for high-affinity uptake of all branched-chain amino acids or that different high-affinity systems have a structural component in common. Another hypothesis is that high-affinity uptake of branched-chain amino acids, as identified in the wild type, is completely absent in strain C2901, revealing one or several weak superimposed systems already present in the wild type. According to this hypothesis, the phenotype of the bapl strain could be caused by (1) a regulatory mutation, (2) an inactivating mutation in a structural gene for a common permease or (3) a defect in a gene for a structural component common to two or more different high-affinity permeases. It is unlikely that the mutation affects membrane potential, electrochemical proton gradient, etc., as low-affinity uptake of L-isoleucine and L-leucine as well as uptake of L-citrullineappear to be unaffected. Moreover, the growth rate for the mutant in the absence of SM is the same as for the wild type (data not shown). The assumption of one common carrier for highaffinity uptake of L-valine,L-isoleucineand L-leucine in the wild type is in agreement with most of the data for inhibition by various amino acids at 10 mM. The branched-chain amino acids as well as the related amino acids L-alanine and L-cysteine inhibit in all cases. Moreover, in most cases the degree of inhibition can be explained by the affinities of the putative high-affinity permease for the branchedchain amino acids. However, the poor inhibition of L-leucine uptake by L-isoleucine and vice versa is more difficult to explain from the hypothesis of one high-affinity carrier. The possibility that the lowaffinity component for L-isoleucine transport in the wild type cannot be inhibited by L-leucine has been considered, because this system contributes 25% to the total uptake at a L-leucine concentration of 0.2 mM. However, inhibition of L-isoleucine uptake
S. TULLIN ETAL.
in the bapl mutant, which is assumed only to contain this system, argues against this interpretation, since unlabelled L-isoleucine and L-leucine in this case inhibit to a similar extent. Two or more binding sites on the putative branched-chain amino acid permease for its substrates may allow various explanations for the poor inhibition of L-leucine uptake by L-isoleucine and vice versa. The existence of two closely related forms of the BAPl-dependent system, for example different in conformation or composition, is a rather straightforward hypothesis that could explain these data. This hypothesis could also encompass the finding that bapl is fully recessive. The competition data and the structural similarity between L-alanine, L-cysteine and the branched-chain amino acids point to the possibility that L-alanine and L-cysteine could be substrates for the putative branched-chain amino acid permease (BAP). This has not been investigated. L-citrulline uptake is unaffected in the bapl mutant, indicating that the GAP is not involved in high-affinity transport of L-leucine in strain S288C under our conditions. Moreover, L-citrulline is not able to compete with uptake of any of the branchedchain amino acids in the mutant, suggesting that the GAP is also not involved in the low-affinity transport. In a study on L-leucine uptake in various strains, Bussey and Umbarger (1970a,b) found that one strain contained a system with a KT of 30 PM,whereas another contained a system with a KT of 1 mM. It is possible that this reflects a polymorphism for a defect in the BAPI-dependent system. The simplest interpretation of the data presented in this paper is to assume the existence of a highaffinity branched-chain amino acid permease in strain S288C. This carrier is completely or partially absent in the bapl mutant.
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ACKNOWLEDGEMENTS We are grateful to Dr S. Carl Falco for supplying sulfometuron methyl. Professor Diter von Wettstein is thanked for support and encouragement. Dr Torsten Nilsson-Tillgren and Dr Steen Holmberg are acknowledged for fruitful discussions, Ms Nina Rasmussen for drawing the figures and Ms Hanne Frederiksen and Ms Gitte Bank for technical assistance. REFERENCES Bussey, H. and Umbarger, H. E. (1970a). Biosynthesis of the branched-chain amino acids in yeast: a leucine-
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
binding component and regulation of leucine uptake. J . Bacteriol. 103,277-285. Bussey, H . and Umbarger, H. E. (1970b). Biosynthesis of the branched-chain amino acids in yeast: a trifluoroleucine-resistant mutant with altered regulation ofleucine uptake. J . Bacteriol. 103,286-294. Cartwright, P. C., Rose, A. H., Calderbank, J. and Keenan, M. H. J. (1989). Solute transport. In Rose, A. H. and Harrison, J. S. (Eds.), The Yeasts, vol. 3, 2nd edn, Metabolism and Physiology of Yeasts. Academic Press, pp. 5-56. Falco, S. C. and Dumas, K. S. (1985). Genetic analysis of mutants of Saccharomyces cerevisiae resistant to the herbicide sulfometuron methyl. Genetics 109,21-35. Garrett, J. M. (1989). Characterization of AATI: a gene involved in the regulation of amino acid transport in Saccharomyces cerevisiae. J . Gen. Microbiol. 135, 2429-2437. Grenson, M., Mousset, M., Wiame, J. M. and Bechet, J. (1966). Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim. Biophys. Acta 127,325-338. Grenson, M. (1966). Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. 11. Evidence for a specific lysine-transporting system. Biochim. Biophys. Acta 127,339-346. Grenson, M., Hou, C. and Grabeel, M. (1970). Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J . Bacteriol. 103,77&777.
94 I
Horak, J. (1986). Amino acid transport in eucaryotic microorganisms. Biochim. Biophys. Acta 864,223-256. Kotliar, N. and Ramos, E. H. (1983). Systems of L-leucine transport into Saccharomyces cerevisiae protoplasts. Biochim. Biophys. Acta 734,378-380. Mortimer, R. K. and Hawthorne, D. C. (1966). Genetic mapping in Saccharomyces. Genetics 53, 165-1 73. Mortimer, R. K. and Johnston, J. R. (1986). Genealogy of principal strains of the yeast genetic stock center. Genetics 113,3543. Ramos, E. H., de Bongioanni, L. C. and Stoppani, A. 0. M. (1980). Kinetics of ~-['~C]leucinetransport in Saccharomyces cerevisiae. Effect of energy coupling inhibitors. Biochim. Biophys. Acta 599,214231. Seaston, A., Inkson, C. and Eddy, A. A. (1973). The absorption of protons with specific amino acids and carbohydrates by yeast. Biochem. J . 134, 1031-1043. Sychrova, H., Horak, J. and Kotyk, A. (1989). Transport of L-leucine in the fission yeast Schizosaccharomyces pombe. Yeast 5,199-207. Tanaka, J.-I. and Fink, G. R. (1985). The histidine permease gene (HIP1) of Saccharomyces cerevisiae. Gene 38,205-2 14. Wainer, S. R., Boveris, A. and Ramos, E. H. (1988). Control of leucine transport in yeast by periplasmic binding proteins. Arch. Biochem. Biophys. 262, 48 1 4 9 0 . Walton, E. F., Carter, B. L. A. and Pringle, J. R. (1979). An enrichment method for temperature-sensitive and auxotrophic mutants of yeast. Mol. Gen. Genet. 171, 1 1 1-1 14.
VOL.7: 933-94 1 (1 99 1)
A High-affinity Uptake System for Branched-chain Amino Acids in Saccharomyces cerevisiae S 0 R E N TULLIN, CLAES GJERMANSEN A N D MORTEN C. KIELLAND-BRANDT* Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received 29 January 1991;accepted 18 June 1991
In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched-chain amino acid uptake. Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics. This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected. Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, Lisoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids. We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild-type allele is denoted BAPI for branched-chain amino acid permease. The BAPI-dependent system is different from the general amino acid permease. KEY WORDS - BAPI; branched-chain amino acids; transport; permease; uptake; L-isoleucine; L-leucine; L-valine; sulfometuron methyl; yeast.
INTRODUCTION Amino acid transport into yeast cells is an active and essentially unidirectional process driven by the electrochemical proton gradient (Seaston et al., 1973; Horak, 1986). The well-characterized general amino acid permease (GAP) is the only general uptake system identified so far in Saccharomyces cerevisiae, whereas a number of specific carriers have been found each responsible for transport of a small group of related amino acids (reviews: Horak, 1986; Cartwright et al., 1989). The high-affinity transport system for L-arginine is an example of a carrier transporting a group of related amino acids, as all three normal basic amino acids and L-canavanine are transported by this system (Grenson etal., 1966; Grenson, 1966). Concerning synthesis and inactivation of carriers, the GAP and the high-affinity L-proline carrier, encoded by the PUT4 gene, are subject to nitrogen*Addressee for correspondence 0749-503X/9 1/090933-O9$05.00 0 1991 by John Wiley & Sons Ltd
catabolite regulation (Cartwright et al., 1989). General amino acid control of carriers has to our knowledge not been published. However, the sequence published for the promoter of the HIP1 gene encoding the histidine permease (Tanaka and Fink, 1985) contains two TGACTC elements. In some cases it has been possible to distinguish kinetically two uptake components. L-leucine uptake, for example, can be explained by two parallel systems, both obeying Michaelis-Menten kinetics (Ramos et d., 1980; Kotliar and Ramos, 1983; Wainer et al., 1988): a high-affinity, low-capacity system and a low-affinity system with high capacity. Studies on amino acid transport in Schizosaccharomyces pombe revealed that L-phenylalanine, Lisoleucine, L-valine and L-cysteine are competitive inhibitors of L-leucine uptake (Sychrova et al., 1989), suggesting that these amino acids are substrates for a L-leucine transporting carrier. The present study describes the identification of a system responsible for high-affinity uptake of Lisoleucine. L-leucine and L-valine.
934 MATERIALS AND METHODS Strains
The parental strain for the mutagenesis was S288C ( M A T a SUC2 ma1 gal2 CUPI). Strain X2180-1A (MATa SUC2 malga12 CUP1) was used as parent in tetrad analysis. Other strains were derived as described. Media
Synthetic minimal medium MA (minimal ammonia) was made by adding 10 g succinic acid, 6 g NaOH, 5 g (NH,)*SO, and 1.7 g Bacto Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco) to 1 litre of H,O. MP is MAmedium containing 2g/l L-proline instead of the (NH,),SO,, Synthetic complete medium, SC, was MA medium supplemented with various nutrients: 20 mg/l adenine sulfate, 20 mg/l L-arginine hydrochloride, 300 mg/l L-aspartic acid, 100 mg/l L-glutamic acid, 20 mg/l L-histidine hydrochloride, 30 mg/l Lisoleucine, 30 mg/l L-leucine, 30 mg/l L-lysine hydrochloride, 60 mg/l L-methionine, 50 mg/l Lphenylalanine, 375 mg/l L-serine, 600 mg/l Lthreonine, 20 mg/l L-tryptophan, 30 mg/l L-tyrosine, 20 mg/l uracil and 30 mg/l L-valine. SC-Val-Ile-Leu was SC medium without L-valine,L-isoleucineand Lleucine. Complex medium with glucose was YPD YO Difco Bacto Yeast Extract, 2% Difco Bacto Peptone, 2% glucose). YPG contained 1% Difco Bacto Yeast Extract, 2% Difco Bacto Peptone, 3% glycerol. YPD+ SM was YPD medium containing Sulfometuron Methyl (N-[(4,6-dimethylpyrimidin2 - yl) - aminocarbonyl] - 2 - methoxycarbonylbenzenesulfonamide, 30 mg/l or 75 mg/l), kindly provided by S. C. Falco, E. I. Du Pont de Nemours & Company, Wilmington, Delaware). All solid media contained 2% Difco Bacto Agar. Mutagenesis and isolation of mutants sensitive to sulfometuron methyl on YPD
Cells of strain S288C were grown in liquid YPD to early stationary phase. Ultraviolet (UV) muta-
genesis was carried out either by plating the cells on YPG and irradiating them in situ or by irradiation of 2 x lo7cells, suspended in 20 ml water in a Petri dish, under agitation. In the latter case, enrichment for SM-sensitive cells was performed by heat killing of exponentially growing cells according to Walton et al. (1979): The cells were harvested, resuspended in 1 ml YPD and grown overnight in darkness to
S. TULLIN ET AL.
stationary phase. After 100-fold dilution in YPD + SM (30 mg/l), cells were grown for 5 h at 37°C (log phase) and heated to 50°C for 7 min. In pilot experiments in YPD, this treatment killed 99.6% of logarithmically growing cells, while 63% of stationary-phase cells survived. Cells were plated on YPG to give 200 colonies/plate. SM-sensitive mutants were identified by replica plating to YPD SM (30 mg/l).
+
Tetrad analysis Tetrad analysis was performed as described by Mortimer and Hawthorne (1966). Mutant and wild type were distinguished by replica plating onto YPD SM (75 mg/l) and reading of the plates after 1 day at 30°C.
+
Uptake assay In all experiments, exponentially growing cells were harvested and assayed for their uptake of a I4Clabelled amino acid. For most experiments an overnight culture in SC medium was diluted into the same medium and grown at 30°C for 5-6 h until an OD,, of 0.5 was attained. Uptake kinetics were assayed in SC-Ile-Leu-Val medium, while inhibition of branched-chain amino acid uptake by a non-labelled amino acid was ma y e d in MA medium. Exponentially growing cells were harvested by centrifugation, washed and resuspended in the medium indicated above. Two hundred microlitres of cells (- 2 mg dry wei ht) were added to 300 p1 medium containing the ',Clabelled amino acid and incubated at 25°C. After 45 min, the assay was stopped by the addition of 900 p1 of an ice-cold 15&250 mM solution of the same amino acid in unlabelled form. The cells were harvested after 0-5 min by centrifugation in a microfuge, washed three times with cold SC medium and counted. Uptake rates are expressed relatively to cell dry weight obtained by drying at 104°C for 18 h. The data presented are the average of three experiments; the average variation was less than 5%. In the experiments where uptake was assayed as a function of extracellular substrate concentration, 16 ~ different concentrations in the range of 2 5 p to 8 mM were used. All radioactive compounds were obtained from New England Nuclear: ~-[ureido-'~C]citrulline (NEC-214), ~-[~~C(U)]isoleucine (NEC-278E), L['4C(U)]leucine (NEC-279E) and ~-['~C(U)]valine (NEC-29 1E).
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
Analysis of the kinetic experiments was carried out using the Enzfitter program distributed by Elsevier BIOSOFT, 68 Hills Road, Cambridge, UK. Proportional weighing was used, i.e. the standard deviation of the determination of uptake rate
J=
935
was taken to be proportional to the uptake rate. Curves in the Eadie-Hofstee presentations (Jversus J / [ S ] ,where J is the uptake rate and [S] is the extracellular substrate concentration) were drawn by the Enzfitter program according to the equation:
J J -KTI--KT2-+JM,+JM2+ PI [SI 2
which was derived by adding two Michaelis-Menten equations and solving for J as a function of J / [ S ] .In the equation JMland KTIare capacity and affinity, respectively, for system 1 , and J M 2and KT2 are capacity and affinity, respectively, for system 2.
because only few generations of growth took place before plating.
Transport assay The uptake assay was designed for accuracy of sampling time. Transport of the labelled amino acid was stopped by the addition of an ice-cold solution Ammonia repression of the GAP of the same amino acid in unlabelled form. Control For the determination of ammonia repression of experiments with I4C-labelledL-valine, L-isoleucine the GAP (Garrett, 1989), an overnight culture in SC or L-leucine in strain S288C show that this approach medium was washed, diluted and transferred to MP efficiently blocks influx for at least 1 h (data not or MA medium. The cells were grown at 30°C for 5- shown). In addition, no detectable efflux can be 6 h until an OD,,, of 0.5 was attained. GAP activity seen. Washing of a large number of samples can was assayed by L-citrulline uptake (Grenson et al., therefore be carried out simultaneously. Branched1970) in fresh MA or MP medium. chain amino acid accumulation in strain S288C was tested for linearity in time. It was linear for at least 10 min (data not shown). RESULTS Uptake was assayed in defined media (SC, MA or Isolation of mutants with impaired uptake of the MP). The intention was to study uptake from a branched-chain amino acids medium of a composition close to that in which the Sulfometuron methyl (SM) impedes growth of S. cells had been growing. In the experiments where cerevisiue wild-type strains on medium lacking L- influx was assayed as a function of external subisoleucine and/or L-valine by inhibiting the aceto- strate concentration (Figures 1-3), SC-Val-Ile-Leu hydroxyacid synthase, encoded by the ILV2 gene was chosen as assay medium. Transport of the (Falco and Dumas, 1985). In contrast, SM has little branched-chain amino acids through carriers with effect when added to complete or complex medium, preference for amino acids other than L-valine, Lbecause most yeast strains can utilize branched- isoleucine and L-leucine should then be reduced to a chain amino acids present in the medium and are as minimum. A disadvantage is that the KT values prea consequence not dependent on de novo synthesis of sented in Table 1 are apparent values because of L-isoleucine and L-valine. In order to isolate possible competitive inhibition by other amino mutants with impaired uptake of either amino acid, acids. However, as will be shown later, the BAP1UV-induced mutants sensitive to SM on complex dependent system is virtually unaffected by the medium were isolated as described in Materials and presence of the additional amino acids. Methods. In the first experiment, cells were plated on YPG and UV-irradiated to 15% survival. By The BAPl gene replica plating of 10000 colonies to YPD+SM, Ten mutants, isolated as described above, were four mutants sensitive to SM were isolated, includ- assayed for their ability to accumulate brancheding C2901 to be described below. In another chain amino acids (data not shown). Three mutants, experiment, where thermal selection followed muta- including one called C2901, were identified with genesis, 14 SM-sensitive mutants were found among impaired uptake of all three branched-chain amino 4000 colonies. Even in the latter experiments the acids. None of the other seven mutants was affected mutants could be considered as being independent, in the uptake of any of the three amino acids, i.e.
936
S. TULLIN ET AL.
1 [VALINE] (mM)
0
200
400
600
800
2
3
4
5
6
[ISOLEUCINE] (mM)
1000
1200
J, / [VALINE] (nmolxgcelts*l. min-l* mM-')
J,l[ISOLEUCINE](nmol =9cell;1"min-1'mM-1)
Figure 2. (a) Influx of ~-isoleucine(J,)as a function of the external ~-isoleucineconcentration in strain S288C (+) and strain C2901(0). Assay medium was SC-Val-Ile-Leu.(b) EadieHofstee plot of the data given in (a) for strain S288C (+) and strain C2901 (0).
Figure 1. (a) Influx of ~-valine(J,)as a function of the external L-valine concentration in strain S288C (+) and strain C2901 (0). Assay medium was SC-Val-Ile-Leu.(b) Eadie-Hofsteeplot of the data given in (a) for strain S288C. (c) Eadie-Hofsteeplot of the data given in (a) for strain C2901.
we found no mutant defective for transport of only one or two branched-chain amino acids. For the primary genetic characterization, auxotrophic markers were avoided. Upon sporulation of the diploid X2180-1A x C2901, a 2:2 segregation of sensitivity to resistance to SM (75mg/l) on YPD was observed in 19 of 20 tested tetrads. Moreover, impaired uptake of branched-chain amino acids cosegregated with SM-sensitivity in four tested tetrads (data not shown). These findings indicate that a single Mendelian gene is affected in the mutant C2901. The wild-type gene is denoted BAPl
(branched-chain amino acid permease). The segregation of SM resistance turned out to be complicated in non-isogenic crosses (data not shown). Uptake of radioactively labelled isoleucine, valine and leucine in the diploids C3391 (BAPIIBAPZ), C3392 (BAPllbapl) and C3393 (bapllbapl) was compared. Table 1 shows that the mutant gene bapl is recessive. In contrast, bapl showed a semidominant phenotype on YPD+SM plates. It may be noted that the wild-type diploid C3391 has a lower uptake relative to cell mass than the wild-type haploids S288C and X2180-1A. Kinetics of L-valine,L-isoleucineand L-leucineuptake The kinetics were investigated in mutant strain C2901 and the parental strain S288C (Figures 1-3; Table 2). A straight line in an Eadie-Hofstee representation of the data is consistent with the presence of a single system obeying Michaelis-Menten kinetics. In the case of a curvature in the EadieHofstee representation of the experimental data, the kinetic parameters giving the best fitting curve for two systems in a J vs J/[S]plot were obtained using the Enzfitter program. In all those cases (Figures 2
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
o
i
z
i
4
i
k
i
i
[LEUCINE] (mM)
- , 0
I
100
200
J, /[LEUCINE]
0
100 J, I
-
400
300
500
(nmol=gce~l;'- min". m W ' )
200
[LEUCINE] (nmol.gcell;'
300
400
= min-', mM-')
Figure 3. (a) Influx of L-leucine (J,)as a function of the external L-leucine concentration in strain S288C (+) and strain C2901 (0). Assay medium was SC-Val-Ile-Leu. (b) Eadie-Hofstee plot ofthe data given in (a) for strain S288C. (c) Eadie-Hofstee plot of the data given in (a) for strain C2901.
and 3), the curves coincided with the experimental data, i.e. the observed kinetics could be explained by two parallel systems obeying Michaelis-Menten kinetics. Figures la, 2a and 3a show that accumulation of all three branched-chain amino acids is affected in the mutant, the effect being most pronounced for Lvaline. Eadie-Hofstee representation shows that Lvaline uptake in the mutant and the wild type fits a model of one system obeying Michaelis-Menten kinetics (Figures Ib and lc). The non-linear EadieHofstee representation of the L-isoleucine influx
937
data for the wild type and the close fitting curve (Figure 2b) obtained with the equation for two systems with the kinetic parameters presented in Table 2 suggest that L-isoleucine uptake is mediated by two systems obeying Michaelis-Menten kinetics in the wild type. In the mutant, on the other hand, Lisoleucine accumulation can be explained by one system obeying Michaelis-Menten kinetics. The system remaining in the mutant has the same kinetic parameters as the low-affinity system identified in the wild type (Figure 2b, Table 2). L-leucine accumulation is consistent with two systems, each obeying Michaelis-Menten kinetics, in both the mutant and the wild type. Free diffusion or a system with very high KT is responsible for the low-affinity component common to the wild type and the mutant (Figures 3b,c; Table 1). Since the KT values for this (these) low-affinity system(s) obtained from the computer anaylsis were larger than 1 M, the diffusion constant ( D = J M K Tis ) presented in Table 1, rather than the estimated KT and JMvalues. There are two obvious alternative interpretations of the data for L-valine accumulation. The carrier identified in the wild type is absent in the mutant, leaving a system which was too weak to be detected when superimposed with the stronger system, or the transport system in the mutant is a mutated version of the wild-type carrier with reduced activity. The kinetic parameters presented in Table 1 reveal a diffusion or very low-affinity component of Lleucine uptake, which is identical in the mutant and the wild type. The medium-affinity system in the mutant appears to be a mutated version of the highaffinity carrier found in the wild type. However, it cannot be excluded that it is a third system, masked in the wild type by the other systems. Inhibition of L-valine, L-isoleucine and L-leucine uptake by various amino acids Uptake of L-valine, L-isoleucine and L-leucine at 0.2 mM was assayed in the presence of various amino acids at 10 mM in strains S288C and C2901 (Table 3). The absolute increase in the uptake of each amino acid when the uptake from SC-Val-Ile-Leu (Figures 1-3) iscompared to that from SD (footnote of Table 3), is almost the same in the mutant and the wild type. This indicates that the amino acids in SCVal-Ile-Leu are not inhibiting uptake through the BAPI-dependent system and that the low-affinity carrier(s) for the branched-chain amino acids may be rather non-specific.
938
S. TULLIN ET AL.
Table 1. Uptake from SC medium of 14C-labelledL-valine, L-isoleucine and L-leucine (nmol gcells-l min-I). Average of two independent experiments.The average deviation was less than 5%
Genotype
Strain S228C X2 180-1A C290 1 C3003 c 3 137 C339 1 C3392 c3393
Source/reference
MATa M A Ta MATa bapl MATaIMA TaBAPllbapl MATa bapl MATaIMATa BAPIIBAPI MATaIMATa BAPljbapl MATaIMATa bupllbapl
Uptake of Val Ile Leu
Mortimer and Johnston (1986) Mortimer and Johnston (1986) Mutant of S288C, this study X2 180-1A x C2901 Spore clone of C3003
121 283 103 245 15 29 65 155 12 25 79 147 66 155 16 25
S288C x X2180-1A S288C x C3137 C2901 x C3137
550 500 88 373 47 387 347 56
Table 2. Apparent kinetic parameters of L-valine, L-isoleucine and L-leucine uptake in synthetic growth medium in strain S288C and strain C2901 calculated from the data presented in Figures 1-3. KT values are expressed in mM, JMvalues in nmol g-l min-l and the diffusion constant D in nmol g-l min-' mM-l Strain S288C(BAPI) C2901 (bapl) S288C (BAPI) C2901 (bapl) S288C (BAPI) C2901 (bapl)
Uptakeof Val Val Ile Ile Leu Leu
D
KTI
JMl
KTz
JMz
2.0 7.2
2400 950
-
-
-
-
-
-
0.3 1 4.2
540 2000
-
0.16 0.94
970 360
Uptake in the wild type in the presence of various amino acids (Table 3 ) reveals similar inhibition patterns for L-valine, L-isoleucine and L-leucine transport. L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine inhibit in all cases, to various extents. These findings suggest that highaffinity uptake of branched-chain amino acids in the wild type is carried out by one carrier or by several carriers with similar inhibition patterns. Moreover, L-methionine inhibits L-valine uptake to 38%, while L-glycine does not inhibit in any case. Assuming one high-affinity carrier with the kinetic parameters for L-valine, L-isoleucine and L-leucine uptake as presented in Table 2 (KT, and J M ,for strain S288C), Lisoleucine and L-leucine should be good competitors of L-valine transport. On the other hand, only weak inhibition of L-isoleucine transport and in particular L-leucine transport by L-valine is expected. These assumptions fit the data. However, the assumption causes difficulties in explaining why uptake of L[I4C]isoleucine in the presence of excess unlabelled
11
3900
-
-
-
-
-
-
-
54 53
L-isoleucine at 10 mM is about ten-fold lower than the uptake in the presence of L-leucine at 10 mM and vice versa (Table 3). In strain C2901, the competitors investigated have weak and similar effects, suggesting that the system(s) responsible for branched-chain amino acid uptake in the mutant are relatively unspecific, low-affinity system(s).
G A P repression in the bapl mutant C2901 Recently, a new amino acid uptake mutant designated aatl was described (Garrett, 1989). When combined with a leu2 allele, aatl makes yeast unable to grow on rich medium or minimal medium supplemented with certain amino acids. Moreover, GAP activity was not repressed by ammonia in the mutant. It is an obvious possibility that aatl mutants are sensitive to SM on complex medium. To determine whether bapl has an aatl-like phenotype, ammonia
939
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
Table 3. Uptake of ''C-labelled L-valine, L-isoleucine or L-leucine at 0.2 mM in the presence of various non-radioactive amino acids at 10 mM. Numbers represent the rate of uptake as a per cent of the uptake in the absence of competitor
S288C ( B A P I ) Potential competitor None Ala Arg ASP CYS Glu GlY His Ile Leu LYS Met Phe Pro Ser Thr Trp Val Citt
Val 1oo*
4.8 90 119 20 74 104 87 7.0 5.8 83 38 141 86 94 76 36 7.0 ND
Uptake of Ile
loo* 18 100 112 54 98 89 98 3.8 30 112 103 96 87 89 80 64 47 ND
C2901 (bupl)
Leu
Val
Uptake of Ile
Leu
1 oo* 26 93 91 59 81 82 77 48 4.8 64 123 149 126 98 79 68 59 ND
loo* 21 ND ND 52 ND ND ND 21 20 ND 31 ND ND ND ND ND 34 102
loo* 37 ND ND 68 ND ND ND 39 52 ND 60 ND ND ND ND ND 42 90
1 oo* 21 ND ND 85 ND ND ND 26 12 ND 42 ND ND ND ND ND 58 113
*Absolute rates of uptake in this row, i.e. in the absence of competitor, were 300, 400,750,75,200 and 250nmol g - ' min-', respectively. tL-ci trulline. ND, not determined.
repression of the GAP in strains S288C and C2901 was investigated. Since L-citrulline is known to enter the cell solely through the GAP (Grenson et af., 1970), L-citrulline uptake was assayed after growth in MP and MA medium for 5 h. Table 4 shows that ammonia repression of GAP activity in the bapl mutant is identical to the repression seen in the wild type, suggesting that bapl is different from the aatl mutation.
DISCUSSION The present investigation defines a Mendelian gene, BAP1, by a mutation resulting in sensitivity to SM on the complex medium YPD. The bapl mutation causes a defect in high-affinity uptake of the branched-chain amino acids. The bapl mutation is distinct from the aatl mutation described by Garrett (1989).
Table 4. L-citrulline uptake (nmol g-' min-.') a t 0.1 mM in strains C2901 and S288C. Cells were grown in either MA or MP medium for 5 h and assayed for the uptake of L-citrulline Nitrogen source Ammonia Proline
S288C ( B A P 1 )
C2901 (bapl)
140 5200
160 4900
The kinetic parameters in Table 2 suggest that the low-affinity component for L-leucine uptake in the bapl mutant is the same as the low-affinity system in the wild type. Also, the kinetic parameters for the single uptake system for L-isoleucine in the mutant are similar to those for the low-affinity system in the wild type, suggesting that they are identical systems. This implies that the bapl mutation does not affect
940 low-affinity uptake of L-leucine and L-isoleucine.On the basis of this interpretation, the impaired uptake of branched-chain amino acids in the bapl mutant can be explained in two ways, with overlapping implications. One possibility is that a mutant version of a bapldependent system is responsible for L-valine uptake in the mutant and for medium-affinity L-leucine uptake in the mutant. A corresponding change in the system for high-affinity L-isoleucine uptake might well reduce transport to a level where identification would be difficult as a consequence of superposition with the low-affinity system. Altogether, this hypothesis implies that one permease is responsible for high-affinity uptake of all branched-chain amino acids or that different high-affinity systems have a structural component in common. Another hypothesis is that high-affinity uptake of branched-chain amino acids, as identified in the wild type, is completely absent in strain C2901, revealing one or several weak superimposed systems already present in the wild type. According to this hypothesis, the phenotype of the bapl strain could be caused by (1) a regulatory mutation, (2) an inactivating mutation in a structural gene for a common permease or (3) a defect in a gene for a structural component common to two or more different high-affinity permeases. It is unlikely that the mutation affects membrane potential, electrochemical proton gradient, etc., as low-affinity uptake of L-isoleucine and L-leucine as well as uptake of L-citrullineappear to be unaffected. Moreover, the growth rate for the mutant in the absence of SM is the same as for the wild type (data not shown). The assumption of one common carrier for highaffinity uptake of L-valine,L-isoleucineand L-leucine in the wild type is in agreement with most of the data for inhibition by various amino acids at 10 mM. The branched-chain amino acids as well as the related amino acids L-alanine and L-cysteine inhibit in all cases. Moreover, in most cases the degree of inhibition can be explained by the affinities of the putative high-affinity permease for the branchedchain amino acids. However, the poor inhibition of L-leucine uptake by L-isoleucine and vice versa is more difficult to explain from the hypothesis of one high-affinity carrier. The possibility that the lowaffinity component for L-isoleucine transport in the wild type cannot be inhibited by L-leucine has been considered, because this system contributes 25% to the total uptake at a L-leucine concentration of 0.2 mM. However, inhibition of L-isoleucine uptake
S. TULLIN ETAL.
in the bapl mutant, which is assumed only to contain this system, argues against this interpretation, since unlabelled L-isoleucine and L-leucine in this case inhibit to a similar extent. Two or more binding sites on the putative branched-chain amino acid permease for its substrates may allow various explanations for the poor inhibition of L-leucine uptake by L-isoleucine and vice versa. The existence of two closely related forms of the BAPl-dependent system, for example different in conformation or composition, is a rather straightforward hypothesis that could explain these data. This hypothesis could also encompass the finding that bapl is fully recessive. The competition data and the structural similarity between L-alanine, L-cysteine and the branched-chain amino acids point to the possibility that L-alanine and L-cysteine could be substrates for the putative branched-chain amino acid permease (BAP). This has not been investigated. L-citrulline uptake is unaffected in the bapl mutant, indicating that the GAP is not involved in high-affinity transport of L-leucine in strain S288C under our conditions. Moreover, L-citrulline is not able to compete with uptake of any of the branchedchain amino acids in the mutant, suggesting that the GAP is also not involved in the low-affinity transport. In a study on L-leucine uptake in various strains, Bussey and Umbarger (1970a,b) found that one strain contained a system with a KT of 30 PM,whereas another contained a system with a KT of 1 mM. It is possible that this reflects a polymorphism for a defect in the BAPI-dependent system. The simplest interpretation of the data presented in this paper is to assume the existence of a highaffinity branched-chain amino acid permease in strain S288C. This carrier is completely or partially absent in the bapl mutant.
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ACKNOWLEDGEMENTS We are grateful to Dr S. Carl Falco for supplying sulfometuron methyl. Professor Diter von Wettstein is thanked for support and encouragement. Dr Torsten Nilsson-Tillgren and Dr Steen Holmberg are acknowledged for fruitful discussions, Ms Nina Rasmussen for drawing the figures and Ms Hanne Frederiksen and Ms Gitte Bank for technical assistance. REFERENCES Bussey, H. and Umbarger, H. E. (1970a). Biosynthesis of the branched-chain amino acids in yeast: a leucine-
HIGH-AFFINITY UPTAKE SYSTEM FOR BRANCHED-CHAIN AMINO ACIDS
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