JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1225-1238 Copyright © 1976 American Society for Microbiology

Vol. 127, No. 3 Printed in U.S.A.

Regulation of Branched-Chain Amino Acid Transport in Escherichia coli DALE L. OXENDER* Department ofBiological Chemistry, The University of Michigan Medical School, Ann Arbor, STEVEN C. QUAY

AND

Michigan 48109 Received for publication 18 May 1976

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyltransfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.

The regulation of the biosynthesis of leucine, isoleucine, and valine has been studied in detail by Umbarger and co-workers (for review, see references 53-55). These extensive studies have led to considerable insight into the complex mechanisms for the regulation of enzyme activity and synthesis, including allosteric effects (56) and multivalent repression (16). In contrast, the regulation of branched-chain amino acid transport has received much less attention. A transport system for leucine, isoleucine, and valine is repressible by leucine but not isoleucine and valine (39, 41), although other studies have implicated the latter two amino acids (50-51) as well as cysteine (27), methionine, and alanine (21-22, 50-51). A regulatory system that involves both cognate (leucine, isoleucine, and valine) and noncognate (cysteine, methionine, and alanine) amino acids would be very unusual and seemed unduly complex. We therefore felt that a detailed study of the regulation of transport was justified. Preliminary work indicated that transport was not coordinately regulated with leucine, isoleucine, or valine biosynthesis (43), although both processes are regulated by an interaction involving the leucyl-transfer ribonucleic acid (tRNA) synthetase and its products (31, 42). In this communication, the parameters important for both repression and derepression of leucine,

isoleucine, and valine transport are examined. (Part of the work presented here was submitted by S.C.Q. in partial fulfillment of the requirements for the Ph.D. degree from the University of Michigan, Ann Arbor.) MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. Growth supplements for auxotrophic strains when not indicated in text were (in millimolar concentrations): uracil, 0.4; thymine, 0.3; leucine, 0.4; isoleucine, 0.4; valine, 1.0; arginine, 0.2; histidine, 0.2; and methionine, 0.5. For all experiments, the medium consisted of the morpholinopropane sulfonate-N-tris(hydroxymethyl)methyl glycine-buffered salts solution (designated MOPS) described by Neidhardt et al. (35). The details of media preparation were as described in that paper. Minimal medium was prepared by adding 0.4% (wt/vol) D-glucose to MOPS (MOPS-G). All solutions of supplementary nutrients were sterilized by filtration through a 0.2-,um membrane filter (Nalgene filter unit, Nalge Sybron Corp., Rochester, N.Y.). Distilled water was sterilized by autoclave. Cultures (50 ml) for enzyme or transport assays were grown aerobically in 250-ml Erlenmeyer flasks in a shaking water bath (New Brunswick Scientific Co., model G-76) that maintained a constant temperature between 30 and 41 + 0.250C. The platform rotation was approximately 150 rpm. For the isola-

1225

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QUAY AND OXENDER

J. BACTERIOL.

TABLE 1. List of E. coli strainsa Strain

Genotype°

CU5 CU361 CU367

met gal rbs leu-455 gal ilvC462 leu-455

CU372

gal ilvADAC115 leu-455

CU1018

ilvS505 ilvT504 ilvU506

ELK4

pyrAl ara-leuA101 str, B/r

E0115 E0300 E0302 KL231

ilv, W3110 Wild-type K-12, Fleu thy-35 str-120 leuS31

Source or comment

H. E. Umbarger H. E. Umbarger H. E. Umbarger; presumptive single-site ilvC mutation H. E. Umbarger; deletion mutant of ilvA and parts of ilvD and ilvC H. E. Umbarger; isoleucyl-tRNA synthetase mutant E. L. Kline; deletion mutant from araABOC and including leuDCBAO genes Laboratory stock ATCC 14948r via R. L. Somerville

J. J. Anderson; temperature-sensitive leucyltRNA synthetase mutant NP29 pyr valSI, KB J. J. Anderson; temperature-sensitive valyltRNA synthetase mutant a All strains except ELK4 and NP29 were derived from E. coli K-12. bFor gene symbols, see Taylor and Trotter (52). ' ATCC; American Type Culture Collection. tion of shock fluid for assaying binding proteins, 1liter cultures were grown aerobically in Fernbach flasks. The growth rates for large and small cultures were indistinguishable. To shift cells from medium of one composition or temperature to another, the culture was rapidly chilled and centrifuged aseptically at 10,000 x g for 10 min, and the cells were resuspended in prewarmed medium of the desired composition. All experiments reported here were performed on cultures growing at densities between 0.004 and 0.30 mg/ml (dry weight). Measurement of growth. Bacterial mass was estimated by measuring the optical density of cultures using a Zeiss PMQ2 spectrophotometer. Only data obtained after at least three generations of growth and between 0.1 and 1.0 absorbancy units at 420 nm (A420 units) (1-cm light path) were used to calculate growth rates. Samples of growing cells were periodically transferred aseptically to 0.9% (vol/vol) formaldehyde to stop growth. These samples were then diluted with water to obtain A420 readings between 0.1 and 0. 4. Growth is expressed as the specific firstorder rate constant (k), in units of hour-', according to the expression, k, = (ln 2)/(mass doubling time). The first-order rate constant, k, was calculated from the slope of the least-squares plot of the natural logarithm of A420 as a function of time, in hours, by the following equation: xj -yi nXy

k= S

Xi2 -r2

where xi is time, yi is the natural log of A420, x is mean of all time values (1/n xi), y is mean of all ln(A420) values (1/n I y;), and n is the number of points in each plot. _

Preparations of cell extracts. Cell-free extracts were prepared by sonic treatment of washed cells as previously described (9). The protein content of extracts was measured colorimetrically at 750 nm by the method of Lowry et al. (32), using bovine serum albumin as standard.

Enzyme assays. Threonine deaminase (EC 4.2.1.6; L-threonine hydrolyase [deaminating]:threonine dehydratase) activity was assayed in cell-free extracts by the method described by Burns (7), except that the absorbance of the 2,4-dinitrophenylhydrazone derivative of a-ketobutyrate was measured at 530 nm in a Zeiss PMQ2 spectrophotometer. a-Isopropylmalate synthetase (EC 4.1.3; a-isopropylmalate-a-keto-isovalerate-lyase [coenzyme A acetylating]) was measured by the method of Kohlhaw and Leary (30). For these enzyme assays, 1 unit of activity is the production of 1 gmol of product in 1 min under the standardized assay conditions. All values reported are averages of at least two determinations with appropriate blanks subtracted. Transport assays. Cultures of cells for transport assays were harvested in exponential phase by centrifugation and washed three times with 4°C MOPS. The cells were resuspended in 37°C MOPS and incubated 5 min at 37°C. The reaction was started by adding the bacterial suspension to a solution of the radioactive amino acids and any other addends as described in the text. The time course of uptake was measured by removing 0.5-ml samples at appropriate intervals, filtering these through 24-mm nitrocellulose filters, 0.45-,um pore size (Millipore Corp., Bedford, Mass.), and washing immediately with 5 ml of 37°C 0.01 M potassium phosphate, pH 7.2, and 0.1 mM MgSO4. Radioactivity from the dried filters was counted in a Packard liquid scintillation spectrometer with a Triton-based scintillator which contained, per liter: 2,5-diphenyloxazole, 8.25 g; 1,4-

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AMINO ACID TRANSPORT REGULATION

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bis[2(4-methyl-5-phenyl-oxazolyl)]benzene, 0.25 g; toluene, 667 ml; and Triton X-100, 333 ml. Tritium was counted with an efficiency of 39%. The quantity of cells was estimated from the A4, by using the empirical relationship derived by Koch (29): w (milligrams/milliliter, dry weight) = 0.1361A + 0.03719(A2). The use ofthis equation has been shown to be unrelated to the growth rate of the cells. For the transport experiments, 1 unit of activity is the uptake of 1 ,umol of substrate in 1 min under these standardized conditions. The kinetics of leucine transport were determined by measuring the transport velocity of leucine at concentrations from 8.3 x 10-8 to 5 x 10-6 M. These data ([leul and v5b,) were plotted by the LineweaverBurk method, and the slope and intercept were determined by a least-squares analysis. For these calculations, the following equations were used:

nlp slope=

1

[leu]I

RESULTS Validity of transport and binding assays. Since a major portion of the data reported in this paper represents the uptake of radiolabeled compounds by bacterial cells in suspension, we wished to establish the parameters that were important for the quantification of this process. Preliminary experiments indicated that, under the conditions examined here, uptake was linear with time for at least 50 s. Our choice of a 20-s time period for our assays, therefore, approximates the measurement of initial rates. The rate of uptake was linearly proportional to the dry weight of cells in the range of 0.11 to 0.26 mg of cells per ml and remained unchanged during the time 1 -I 1 1 , [leu]ivj V.bS i A

1]i)

[leu 1

[Iu]~

-

*-*

[leu]~

A

intercept= and the corresponding standard deviations were obtained:

/t (intercept + slope [leu] - v-)

SD(slope)

~'A

SD(in/ep)S (

j

SD(intercept) = where A = n , (1/[leu] )2 - E (1/[leuI)2, n is the number of data points in the plot, and SD is the standard deviation. The slope and intercept from the two linear portions of the biphasic Lineweaver-Burk plot were then used to calculate two Km and two Vma. values, using the method of Neal (34). Amino acid binding assays. The specific activity of proteins in osmotic shock fluid that bind the branched-chain amino acids was determined by equilibrium dialysis (37), in a microdialysis multichamber device (Hoefer Scientific Instruments, San Francisco, Calif.). A 100-,ul amount of a protein solution containing 1 to 2 mg of protein per ml was dialyzed against 0.7 ml of [3Hlleucine in 10 mM potassium phosphate, pH 7.4, and 10-4 M MgSO4 to give a final concentration of 8.8 ,uM leucine. Dialysis was carried out at 4°C until equilibrium was established. Samples of 30 ,ul were removed, and the radioactive leucine was measured by liquid scintillation spectrometry as described above. One unit of activity represents the binding of 1 jtmol of leucine under these assay conditions. The values reported in this paper represent the mean of three determinations and vary with a standard deviation of 20%. Source of chemicals. All chemicals were obtained from commercial sources and were of reagent grade or better.

(intercept +

slope [l-u, - 1)

A

(about 30 min) required for each experiment. A prospective study over several months of the reproducibility of duplicate or triplicate uptake measurements indicated the transport capacity of cells could be determined with an SD of +3.6% and a standard error of 0.3% (range ±+13%; n = 147). On two occasions, seven cultures of presumably identical cells (strain E0300) were grown and harvested and the transport capacity was determined. For these 14 cultures, the biological variability led to SD values of +5% and + 11% in the determination of transport capacity. These preliminary studies allowed us to assume that the transport capacity of a culture of cells will depend on the remaining undefined variables, the amount of transport components present per cell and the energy reserves for transport. The major cellular energy sources for uptake are metabolic energy and the chemical potential of the intracellular amino acids, which can be used to drive transport by counterflow mechanisms (57). Evidence has been presented elsewhere (41) to

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QUAY AND OXENDER

show that the pool amino acids are not retained when the cells are washed with fluid below approximately 8°C. Ice-chilled buffer was always used to wash cells in preparation for these experiments. Changes in the level of intrinsic metabolic energy could cause changes in the apparent transport capacity of cells and affect the results presented here. We have in fact demonstrated an increase of about twofold in the rate of uptake after the addition of glucose to the transport assay. Although the data presented in this paper are from unsupplemented cells, the effect of the addition of a metabolizable substrate was tested and found not to vary with the growth conditions or with the physiological states studied in this paper. Branched-chain amino acid-binding proteins appear to be the rate-limiting component for transport as determined by genetic and kinetic experiments (37, 39, 42, 44, 46). Since the total amount of shock fluid protein per quantity of cells did not vary under the conditions tested, an increase in specific activity for the branched-chain amino acid-binding proteins indicates an increase in the differential rate of expression of this protein activity. In all cases, the levels of transport capacity and binding activity were in good agreement. Our calculations indicate that binding of transport substrates to these binding proteins accounted for less than 0.004 nmol per mg of cells (dry weight) during our uptake assays. The above considerations have led us to conclude that differences in transport activity for cells in cultures that are genetically or physiologically distinct can be considered significant if the observed differences in transport activity are greater than 22%, since these would be more than 2 SD apart, as determined for the biological variation above. Regulation of branched-chain amino acid transport in wild-type E. coli. The purpose of these experiments was to investigate the hypothesis that branched-chain amino acids, or some derivative, should be the effectors in any process that might regulate the differential rate of fornation of branched-chain amino acid transport components. Since exogenous leucine has been used extensively to cause repression of branchedchain amino acid uptake (44), we wish to verify and extend this aspect of the regulation of transport. Figure 1 shows a differential plot of the ileucine transport capacity as a function oftotal cellular mass in cultures ofE. coli K-12 grown with or without L-leucine. The time course of the change in transport capacity of

the cells when the leucine content of the culture medium was quickly altered is also depicted. The cultures in steady-state growth indicate that the transport components in wild-type E. coli can be varied over a 16-fold range, from 0.03 U per mg of cellular protein when grown with leucine to 0.48 U per mg of cellular protein when grown in minimal medium. A positive, non-zero intercept was observed for both these cultures, which was subtracted to obtain Fig. 1. The nature of this component (0.014 U/ml for control cells; 0.003 U/ml for leucine-supplemented cells) is not known. Upon removal of leucine, a repressed culture rapidly assumed the rate of transport expression characteristic of minimal mediumgrown cells. The increase in transport capacity upon removal of leucine required active protein synthesis as well as the removal of exogenous leucine, since either chloramphenicol or rifampin added at the time that leucine was removed stopped the subsequent increase (Fig. 2). The addition of leucine to a culture caused the rate of transport expression to return to the rate in leucine-supplemented cultures. There appeared to be no slowing in the expression of transport capacity, as would be expected if leucine stimulated degradation of some protein(s) important for transport. Although bacteria catabolize proteins at a relatively low rate during logarithmic growth, a number of factors, including carbon or nitrogen starvation, can cause this process to in-

0.10

;

w

Y-. a.

w

0D5-

z w

-J CULTURE MASS (mg/mI)

FIG. 1. Differential plot of leucine transport in E. coli wild type as a function of culture mass. Strain E0300 was grown in MOPS-G in steady state with 0.4 mM leucine (open circles) or without leucine (closed circles). At the times indicated, 0.4 mM leucine was added to the minimal medium culture (closed triangles) or the leucine was rapidly withdrawn (open triangles). Leucine transport at 0.5 iM leucine and cell mass were determined as described in Materials and Methods.

VOL. 127, 1976

AMINO ACID TRANSPORT REGULATION

C

I

.E

I

I

a2.0 10

0

w 4

o

a-

J 1.0

A0 0

1229

nation: that growth slows after leucine is added and that this time is needed for minimal medium-grown cells to "adapt" to the presence of leucine before growth can resume. A careful examination of the growth of this culture before and after the addition of leucine supports the latter hypothesis (Fig. 4). A delay of 20 to 30 min was consistently observed when leucine was added to cultures in MOPS-G; the presence of isoleucine and valine prevented this delay. Once unrestricted growth resumed,

A

0

w -J

4:

r

60 80 40 TIME, min FIG. 2. Effect of chloramphenicol or rifampin on the time course of leucine transport derepression. Strain E0300 was grown under repressed conditions, washed, and resuspended in the following media: (closed circles) minimal medium without leucine; (open circles) minimal medium with 200 mg of rifampin per liter; (closed triangles) minimal medium with 200 mg ofchloramphenicol per liter. Samples were withdrawn at indicated intervals and washed, and leucine transport was measured as indicated in Materials and Methods. 20

TIME, (min)

severalfold (for review, see reference 19). To test whether leucine stimulated degradation of a transport component, the following experiment was performed. Wild-type E. coli was grown in MOPS-G to mid-log phase, and the culture was divided. To three subcultures

crease

added leucine, leucine and chloramphenicol, or chloramphenicol alone. At various times, samples were withdrawn and transport capacity was tested. If leucine can stimulate degradation of a transport component(s) in the absence of protein synthesis, differences should be apparent between cultures with and without leucine. Over a 100-min period, there was no effect of leucine on the cultures receiving chloramphenicol (Fig. 3). A slow (half time = 2.5 h) loss of transport capacity was seen with these cells, which was unaffected by the presence of leucine. The nature of this loss is not known. The cultures receiving leucine alone exhibited a biphasic response, with a delay of 30 to 40 min before a first-order decay in transport capacity was observed. This delay during the transition to steady-state growth with leucine could represent the time needed to form an active repression complex with leucine. However, observations of leucine sensitivity inE. coli (28, 47) suggest a simpler expla-

was

FIG. 3. Effect of chloramphenicol on the time course of leucine transport repression. Strain E0300 was grown in MOPS-G, and then 0.4 mM leucine was added. This culture was divided and growth continued in: MOPS-G plus leucine (closed triangles); the same medium plus chloramphenicol (closed circles); the same medium without leucine but containing chloramphenicol (open circles). -

E C

O

0.4

w

0.3

z 4

ID

°0.2

4o

20

40 TIME

60

80

(min)

FIG. 4. Effect of leucine addition on the growth of strain E0300. Strain E0300 was established in steady-state growth in MOPS-G. At the time indicated, either 0.4 mM leucine (open circles) or 0.4 mM leucine, 0.4 mM isoleucine, and 1.0 mM valine (closed circles) were added. Growth was followed as described in the Materials and Methods. The data have been corrected for the small (1%) dilution that occurred when the additions were made.

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QUAY AND OXENDER

the transport capacity was lost in a first-order 3) with a rate constant (0.83/h) that observed for the growth of the culture (k = 0.77/h). Templeton and Savageau (50) also observed a first-order loss of transport activity during repression that equaled the growth rate of their cultures. If leucine indeed stimulates degradation, it must also stimulate synthesis to a small extent to give the results observed. A much simpler interpretation is that leucine does not affect degradation but decreases the rate of synthesis of a transport component(s). These preliminary experiments lead us to conclude that exogenous leucine can repress the synthesis of membrane components involved in the transport of leucine by E. coli. The word "repression" has been used here in a descriptive sense to define the change in the differential rate of expression of transport capacity with changes in the growth media and does not imply a particular mechanism or site of action. Derepression of transport. Since common transport systems for leucine, isoleucine, and valine have been proposed by genetic and biochemical studies (39, 44) and since exogenously added leucine, isoleucine, and valine have been implicated in the regulation of transport (51), we wished to test whether these three amino acids together could lower transport capacity by a multivalent repression (16) in a manner analogous to the regulation ofthe ilvADE operon. In the latter case, the presence process (Fig. very close to

J. BACTERIOL.

of all three branched-chain amino acids is required for repression. Table 2 contains the results of experiments to test this hypothesis. Strain CU367 required leucine, isoleucine, or valine for growth in minimal medium and could therefore be selectively limited for a single branched-chain amino acid. When this strain was limited for leucine, a four- to sevenfold increase in the transport of leucine, isoleucine, and valine was observed. A similar derepression of the ilvA gene product, threonine deaminase, was observed. When limitation for isoleucine or valine occurred, the transport activity for all the branched-chain amino acids remained repressed, although the activity of threonine deaminase was increased. Since this enzyme derepresses in response to the limitation of any single branched-chain amino acid (16), it served as an outside indicator of the level ofthese amino acids available to the cells. These results support the hypothesis that the regulation of transport is linked only to the intracellular leucine level. The possibility still existed that the limitation signal generated by isoleucine and valine starvation came later than the leucine starvation signal in these non-steady-state experiments and that a depression of threonine deaminase but not of transport components could occur before growth ceased. This possibility was tested by establishing cultures in steadystate growth on the glycyl-dipeptide of each

TABLE 2. Regulation of transport and biosynthesis during branched-chain amino acid limitation Growth Trnportb (U/g of cells dry wt) Leucine- Threorate conmine Strain Growth conditionsa binding stha-nt Leucine IscolneuValine deamgiactivitye act cine (h-') sety CU367 (ilvC426 Nutrient broth 1.35 0.11 0.11 0.05 19 leu455) Supplemented 0.74 0.09 0.06 0.06 0.42 22 Limiting leucine 0.63 0.37 0.24 1.35 147 Limiting isoleucine 0.08 0.07 0.03 0.46 206 0.08 Limiting valine 0.09 0.05 0.38 149 Glycyl-leucine 0.39 0.24 0.15 0.13 Glycyl-isoleucine 0.32 0.09 0.05 0.06 Glycyl-valine 0.32 0.09 0.05 0.05 E0115 (ilv)

Isoleucine and valine 0.52 0.10 0.13 40 111 Limiting isoleucine 0.52 0.12 0.17 Limiting valine 1.24 0.24 0.29 125 a Supplemented cell cultures contained MOPS-G with 0.4 mM leucine, 0.4 mM isoleucine, and 1 mM valine. For limitation experiments, cultures were grown with an excess of two branched-chain amino acids (CU367) or an excess of one (EO115) and a limiting quantity of the indicated amino acid. The limiting concentrations used were 0.02 mM leucine, 0.02 mM isoleucine, or 0.10 mM valine. Growth was followed turbidimetrically until the culture density did not increase (3 to 4 h), and the cells were then harvested. The dipeptides were supplied at 15 mg/liter with an excess of the other two branched-chain amino acids. b Uptake assays contained 1.5 ,uM valine, 0.5 AtM leucine, or isoleucine. Specific activity expressed as units per gram ofcellular protein, as indicated in Materials and Methods.

branched-chain amino acid (steady-state growth is defined as the absence of a change in the first-order growth rate constant for at least three generations). Growth on the dipeptide of each branched-chain amino acid led to restricted growth, as indicated by the reduction in the first-order growth rate constant. These experiments (Table 2) indicate that limitation for leucine but not isoleucine or valine leads to derepression of transport activity and confirm the experiments done under non-steadystate conditions. Analogous derepression of transport capacity can be achieved in a strain with an intact leucine biosynthetic operon by taking advantage of the fact that restriction of growth by limitation for valine necessarily produces a limitation for leucine (16). Strain E0115, an isoleucine and valine auxotroph, was used to test this assumption. A valine limitation had in fact led to a 2.5-fold increase in transport capacity (Table 2). The first enzyme for leucine biosynthesis, a-isopropylmalate synthetase, showed a fourfold increase under the same conditions (data not shown). The specific activity of the leucine-binding proteins in shock fluid increased under leucine limitation but remained repressed when cells were limited for isoleucine or valine (Table 2). The change in the specific activity of leucinebinding protein in shock fluid gave us the first opportunity to examine a gene product of transport regulation. To confirm that the shock fluid from leucine-grown cells did not contain an inhibitor of the binding assay, appropriate mixtures of shock fluid from leucine-limited and leucine-supplemented cultures were assayed for binding activity. The simple additivity of these results (data not shown) indicated that neither shock fluid contained factors that did not bind leucine but that stimulated or inhibited the leucine-binding protein. Since two kinetically distinct saturable uptake systems exist for leucine (44, 59), we wished to determine the quantitative aspects of leucine-limited derepression. Strain CU367 was grown either with excess leucine, isoleucine, and valine or with limiting leucine. Leucine uptake in these cultures was examined over a 58-fold concentration range, from 8.5 x 10-8 to 5 x 10-6 M. The results (Fig. 5) were obtained by assuming that the following equation described the relation of the observed velocity to the leucine concentration: V

V.a,,- [leucine]

Kin

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AMINO ACID TRANSPORT REGULATION

VOL. 127, 1976

+

[leucine]

VmaX2- [leucine]

Km,

+

[leucine]

The kinetic parameters and SD for

re-

12

X

e; 10 E 8 E

E

0

_j

w -

2

4

6

8

10

12

I/EjEUCINEJ, M I FIG. 5. Kinetics of leucine transport in repressed and derepressed E. coli K-12. Strain CU367 (ilvC, leu-455) was grown in MOPS-G with 1 mM valine, 0.4 mM isoleucine, and either 0.4 mM leucine (closed circles) or 0.02 mM leucine (open circles). Cells were harvested and washed, and transport was measured as described in Materials and Methods. The solid line represents the theoretical curve obtained by applying the kinetic parameters to the equation for two saturable systems, as given in the text.

pressed cells were obtained as described in Materials and Methods and were K. = 0.13 ± 0.01 ,uM, Vmaxi = 0.17 + 0.01 U/g; and KM2 = 2.10 + 0.21 ,AM, Vmax, = 0.20 + 0.01 U/g. When the culture was limited for leucine, the K,,,, and Vmax, values changed to 0.14 + 0.01 ,uM and 0.89 + 0.05, respectively, whereas K,6 was 1.2 ± 0.10 ,M and VmaX2 = 0.35 ± 0.01. The decrease in Km. during derepression is statistically significant and has been observed before. The reason for this is not known. Using the assignments of Rahmanian et al. (44), the values of V,m and Vmax6 correspond to LIV-I, whereas K,6 and VmaX2 represent LIV-II. A fivefold increase in LIV-I capacity and a 75% increase in LIV-II were observed, which are similar to values of8-fold and 2.7-fold obtained for strain ML308-225 grown under repressing and nonrepressing conditions (59). These results taken together are most easily interpreted to indicate that leucine, or a derivative, is directly involved in regulation of branched-chain amino acid transport. Although we can not eliminate a role for valine or isoleucine in transport repression, their contribution must be small or occur very late relative to the leucine-limiting signal. Their apparent I

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role in transport regulation in prototrophic strains should now be reassessed. Regulation of transport by growth with exogenous amino acids. Several studies (21-22, 27, 50) indicate that, in addition to leucine, exogenous isoleucine, valine, methionine, cysteine, or alanine can lower branched-chain amino acid transport. We have already demonstrated that derepression of transport does not accompany limitation for isoleucine or valine. Since the branched-chain amino acid uptake systems are not a major route for the accumulation of methionine, cysteine, or alanine, it seemed possible that their role in repression could be indirect, with their primary action being a disruption of normal amino acid metabolism. To complete this study on the regulation of transport, we undertook experiments to reassess these earlier results. The addition of leucine, isoleucine, or methionine to MOPS-G medium in which an E. coli K-12 wild type was growing led to a decrease in the capacity to take up leucine (Table 3). The uptake of histidine and proline was monitored to determine whether general changes in membrane transport capacity occurred during these experiments. These amino acids are accumulated by binding-protein (2) and membranebound (26) transport systems, respectively. Neither alanine nor cysteine had any effect on leucine, histidine, or proline transport. Isoleucine and methionine showed some capacity to repress leucine uptake, as has been reported elsewhere (21, 50), and their failure to effect histidine or proline transport indicates a degree of specificity. It was conceivable that these supplements to the growth media could affect uptake by simple inhibition, if the washing procedure were incomplete and they remained in the uptake assays. This possibility was shown to be unlikely, since the addition of 0.4 mM leucine

to a minimal medium culture 5 min before harvesting had no effect on the transport capacity of these cells after three washes. The transport was reduced by 41% in these cells washed twice. The saturable nature of the growth supplement-dependent decrease in transport capacity is shown for methionine-grown cells in Fig. 6. A threefold decrease was observed for the transport of 0.5 ,uM leucine as the methionine level was increased to 0.4 mM. A similar threefold decrease was observed for the leucine-binding protein from these strains. The repression of the leucine-binding protein and transport by methionine do not appear to be coordinate at low methionine levels. This could be related to the assay conditions, binding activity having been done at saturation, whereas transport was assayed at a nonsaturating level. This discrepancy could also indicate the nonidentity of all regulatory components for the binding protein and the "transport protein complex." Our methionine and isoleucine supplies were tested for contamination with leucine, using a Beckman amino acid analyzer. No leucine was detected, indicating a contamination of less than 0.64%, an amount insufficient to cause the effects seen here. To test whether the repression of transport by exogenous isoleucine, methionine, or alanine is mediated by alterations in amino acid metabolism, two kinds of experiments were performed: (i) the expression of the leu and ilv operons was monitored in a prototroph grown with these supplements; and (ii) the transport capacity of appropriate auxotrophic strains was examined during experiments in which the intracellular amino acid concentrations of these supplements were lowered below that which is normally maintained. Three strains with auxotrophic requirements for leucine or methionine were used in experi-

TABLE 3. Effect on transport systems and biosynthetic enzymes of additions to the growth media of strain

E0300

Minimal medium supplementa (0.4 mM)

Growth rate

consthrate (h-c )

Transport rates° (U/g of dried cells) Leu

His

Pro

a-Isopro- Threonine

pylmalate

synthetasec

deaminasec nae

None .0.77 0.668 1.0 3.1 5 23 Leucine .0.74 0.158 0.9 2.7 1 39 Isoleucine .0.77 0.547 1.0 3.4 23 11 Methionine .0.80 0.407 0.9 3.0 18 7 Alanine .0.65 0.618 0.9 2.9 8 11 Cysteine .0.79 0.691 1.1 2.9 5 a All assays were performed on cells that had been in exponential growth (37°C) for at least five generations. bSubstrate levels for transport were 0.5 ,uM for leucine (Leu), 1.5 ,uM for histidine (His), and 2.0 ,uM for proline (Pro). c Specific activity expressed as units per gram of cellular protein.

AMINO ACID TRANSPORT REGULATION

VOL. 127, 1976

0.2

[METHIONINEJ

mM

FIG. 6. Concentration dependence of methionine repression on leucine transport and binding proteins. Strain E0300 was grown in minimal medium with the indicated levels of methionine. Cells were harvested, and transport of leucine and binding activity were measured as indicated in Materials and Methods.

ments to set the growth rate by limitation for a

single amino acid. Strain CU5, a methionine auxotroph, did not exhibit an increase in leucine transport with methionine limitation at 0.02 mM, although growth ceased after a 40% increase in cellular mass. Excess leucine could still repress transport even during methionine limitation. If repression of transport by methionine is mediated by changes in the leucine biosynthetic enzymes, a mutation in the leucine operon should prevent the methionine response. Strain E0302 contains a point mutation in the leucine operon. When it was limited for leucine under non-steady-state growth conditions, the strain showed a threefold derepression of transport (0.08 to 0.25 U/g of protein). The presence of isoleucine, valine, or methionine in excess did not effect this response (0.23 U/g of protein). Since repression by methionine was reported in an E. coli B/r strain (50), we tested the ability of methionine to repress transport in a B/r strain with deletion of the leucine biosynthetic operon. Again, neither methionine alone nor isoleucine and valine together could prevent the derepression of transport during leucine-limited growth (data not shown). Table 3 contains the specific activity of the leuA gene product, a-isopropylmalate synthetase, and the ilvA gene product, threonine deaminase, of an E. coli wild type grown with supplements of isoleucine, leucine, methionine, or alanine. Examining the leuA activity first, one can see that, with the exception of leucine supplementation, the other supplements led to reciprocal changes in transport capacity and

1233

leucine biosynthesis; i.e., when leucine biosynthesis was derepressed, there was a repression of transport. These results are entirely consistent with the hypothesis that amino acids other than leucine repress transport levels by increasing the leucine biosynthetic capacity. The effect of these supplements on threonine deaminase activity is also shown in Table 3. The derepression by leucine has been observed before (25) and may represent isoleucine limitation by exchange of intracellular isoleucine for leucine. The repression by isoleucine alone and by methionine and alanine was slight and was probably not significant. The non-steady-state experiments involving auxotrophic strains indicated that isoleucine or methionine limitation did not lead to derepression of transport. Furthermore, changes in the level of expression of branched-chain amino acid biosynthetic operons by methionine and isoleucine have been demonstrated. These conclusions support the hypothesis that amino acids other than leucine do not regulate branched-chain amino transport, except to the extent that they alter leucine biosynthesis. Threonine deaminase and transport regulation. The analogous response of the leucine biosynthetic enzymes and transport components to regulation by leucine or a derivative led us to investigate whether these processes were coordinately controlled (43). We concluded that the regulation of transport and biosynthetic enzymes in E. coli and Salmonella typhimurium are not coordinated with respect to cisdominant regulatory elements and that at least some trans-dominant elements are distinct. The ilvA gene product, threonine deaminase, has been implicated as an aporepressor for the ilvADE (11) operon and as a positive regulatory element in the regulation of ilvB and ilvC (10). Most recently, the regulation of the branchedchain aminoacyl-tRNA synthetases has been shown to be altered by mutations in the ilvA gene (28). The central role of threonine deaminase in these processes led us to investigate whether branched-chain amino acid transport systems were regulated by the ilvA gene or its product. The construction of a deletion mutant of the ilvA gene made a direct test of this hypothesis possible. The regulation of transport in isogenic strains with a point mutation in ilvC or a deletion of ilvA and portions of ilvD and ilvC (ilvDAC115) was examined in steady-state growth under repressing or derepressing conditions. Steady-state growth conditions were obtained by the method of Gahr and Nass (18). Table 4 contains the results of these experiments and indicates no major alteration in the

1234

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QUAY AND OXENDER

derepression response of the ilvDAC deletion strain. A slightly lower transport activity and growth rate constant in strain CU372 was consistently found. The essentially normal regulation of transport under both repressed and derepressed conditions indicates that the gene products missing in this strain are essential neither for maintenance of repression nor for derepression. The slightly lower derepressed transport activity in the ilvDAC deletion strain is interesting and may be related to the inability of this strain to increase leucyl-tRNA synthetase production in response to a leucine limitation (11).

Role of aminoacyl-tRNA synthetases in transport regulation. Aminoacyl-tRNA synthetases and their cognate tRNA species have been implicated in the regulation of amino acid biosynthetic enzymes for histidine (5), leucine (31), isoleucine and valine (55), arginine (58), tryptophan (3), and methionine (4). The regula-

tion of transport was tested in strains with mutations in the valyl-, isoleucyl-, and leucyltRNA synthetases to see whether these proteins were effectors in this regulation mechanism. A preliminary report (42) indicated that leucyl-tRNA or its synthetase was required for repression of leucine, isoleucine, and valine transport. Strain NP29 exhibits a temperature-sensitive phenotype that is due to the production of a

temperature-sensitive valyl-tRNA synthetase (13). At 31°C the strain has repressed isoleucine and valine biosynthetic enzymes when grown in excess branched-chain amino acids. When the culture is shifted to 37°C with no change in the composition of the medium, protein synthesis ceases after a small increase in cell mass, valyl-tRNA is partially deacylated, and the expression of operons for biosynthesis of isoleucine and valine is derepressed (13). These results were confirmed here (Table 5) by measur-

TABLE 4. Effect of an ilvA deletion on regulation of transport and biosynthesis

Strain

Pertinent genotype

Limiting amino

acid0

Growth rate constant

(h-')

Leucne transpor

Threonine deaminaseb

CU361

leu-455

None Leucine

0.76 0.37

0.10 ± 0.01 0.57 ± 0.03

31 276

CU367

leu-455 ilvC462

None Leucine

0.75 0.37

0.14 ± 0.02 0.59 ± 0.03

21 290

leu-455 None 0.71 0.10 ± 0.01 2 ilvDAC115 Leucine 0.28 0.44 ± 0.03 2 a Growth media consisted of MOPS-G supplemented with 0.4 mM L-isoleucine and 1 mM L-valine and either 0.4 or 0.015 mM L-leucine. Cells were grown in steady state for at least five generations by maintaining the culture density between 4 and 38 ,ug/ml (dry weight). b Transport and threonine deaminase specific activities were determined as in Table 2. Values represent mean and standard deviation of four separate experiments.

CU372

TABLE 5. Regulation of transport and biosynthesis in branched-chain aminoacyl-tRNA synthetase mutants Strain Strain

~ Pertinent type

Growth conditions Got odtoa

Growth rate constant (h-')

Leucine Ug deaninase sportb tran-(U/g)danns

NP29

valSI

Supplemented, 31°C Supplemented, 37°C

0.38 -C

0.09 0.10

29 158

CU1018

ilvS505 ilvT504 ilvU506

Supplemented Minimal

0.41 0.34

0.13 0.57

235 582

KL231

leuS31

Supplemented, 31°C 0.44 0.18 35 Supplemented, 38°C _d 2.45 293 Supplemented, 38°C Chloramphenicol 0.10 a Cells were grown in MOPS-G medium supplemented with 0.4 mM leucine and isoleucine and 1.0 mM valine. b Uptake of 0.5 uM leucine. Growth ceased after a 26% increase in culture density. d Growth ceased after a 50% increase in culture density.

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AMINO ACID TRANSPORT REGULATION

ing the activity of the ilvA gene product, threonine deaminase. During these same experiments, leucine transport was monitored and was found not to derepress as growth became limited for valyl-tRNA. This is consistent with the earlier finding (Table 2) that limitation for valine alone did not alter transport activity. A strain with an altered form ofthe isoleucyltRNA synthetase has been isolated and contains at least three mutations that lead to derepression of enzymes for the biosynthesis of isoleucine and valine. This strain grows slowly in minimal medium, and this slow growth can be increased by adding isoleucine to the media. The ilv operon in this mutant is refractory to repression by branched-chain amino acids (49). Table 5 contains the growth rate constant, threonine deaminase activity, and leucine uptake activity in this strain. The threonine deaminase activity was very high in cells grown in minimal medium and was only slightly lowered by growth with the branched-chain amino acids. On the other hand, the regulation of transport showed a normal repression response. This confirms our earlier experiments, which indicated that the regulation of transport was independent of cellular isoleucine levels and was probably also independent of the level of tRNA'le charging. Strain KL231 contains a mutation that results in the production of a temperature-sensitive leucyl-tRNA synthetase (31). When this strain was grown in medium supplemented with the branched-chain amino acids at a permissive temperature (31°C), transport for leucine was repressed (Table 5). Shifting the culture to 38°C led to restricted growth, which ceased after a 50% increase in mass. These cells showed a large derepression of transport activity, which was prevented by protein synthesis inhibitors. The present results are similar to those reported earlier (42) for the response of a B/r strain containing a temperature-sensitive leuS gene and a complete deletion of the leucine biosynthetic enzymes. In the present case, a derepression in the enzymes for biosynthesis of leucine (31) accompanied the transport increase, confirming that a deficit for charged tRNAleU had occurred.

DISCUSSION Repression of transport by leucine. The regulation of branched-chain amino acid transport has been examined in E . coli. The experimental observations can be summarized as follows: branched-chain amino acid transport in prototrophic strains could be repressed by exogenous leucine or by amino acids that could alter the

1235

biosynthetic pathways leading to leucine, whereas limitation of appropriate auxotrophs for leucine, isoleucine, valine, or methionine led to derepression of transport only during leucine limitation. These results led us to conclude that limitation for leucine alone is both necessary and sufficient for derepression of transport. Since the LIV-I and LIV-I transport systems served for the accumulation of leucine, isoleucine, and valine, the regulation of these systems by leucine alone seemed paradoxical. At least two hypotheses could explain this situation. (i) The simple regulation pattern of the enteric organism E. coli has been adapted to the feast-famine conditions of the gut, where, it could be argued, the deprivation or surfeit of the three branched-chain amino acids is always coordinate. In such a situation, the coupling of transport regulation to leucine may have been fortuitous. (ii) The cell must regulate the leucine level independent of isoleucine and valine because of the central role played by leucine in various metabolic processes. The latter hypothesis is supported by an examination of some of the leucine-linked metabolic interactions in E. coli. (i) Leucine represses membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH):NADP+ oxidoreductase (R. L. Hanson, personal communication), membranebound proline oxidase (12), cystathionine synthetase (20), and serine hydroxymethyltransferase (20); inhibits aspartokinase III (46); causes growth inhibition after nutritional shiftdown (1); and leads to the accumulation of unique isoaccepting species of leucine, histidine, arginine, valine, and phenylalanine tRNA's (14, 60). (ii) On the other hand, leucine stimulates the activity of lysyl-, methionyl-, and arginyl-tRNA synthetases (24), S-adenosylmethionine synthetase (20), threonine and serine deaminases (38), D-alanine,glycine (dag) transport (J. C. Robbins, Ph.D. thesis, The Univ. of Michigan, Ann Arbor, 1973) and has a sparing effect on the utilization of glycine as a nitrogen source (15) and the utilization of cyclic adenosine monophosphate during catabolite repression (6). Most of these processes are related directly or indirectly to the catabolism or anabolism of amino acids. Fraser and Newman (15) suggested that intracellular leucine levels may serve as a signal for nitrogen scavaging during periods of amino acid imbalance. This is analogous to a similar role played by ppGpp in amino acid imbalance or the function of cyclic adenosine monophosphate to signal a deficiency in energy supplies and identifies leucine as a potential alarmone (48). This important role of leucine would require a careful regulation of

1236

QUAY AND OXENDER

leucine concentrations independent of fluctuations in the other branched-chain amino acids and dependent only on the relative rates of leucine supply and utilization in protein synthesis. The regulation, by leucine alone, of leucine biosynthesis (8), tRNA aminoacylation (33), and transport is in contrast to the multivalent nature of regulation of isoleucine and valine biosynthesis (16) and tRNA aminoacylation (33), and is consistent with this hypothesis. In addition, the leucine-specific transport system (17), which is present at low levels under normal conditions but which can be genetically derepressed (44), may serve as yet another method to control leucine levels independent of isoleucine and valine. In these experiments we have not attempted to separate these alternatives. The experiment depicted in Fig. 1 indicates that removal of exogenous leucine either rapidly lowers the cellular level of some negative component of transport regulation by inactivation or by dilution with new growth, or signals the synthesis of some positive control element in transport regulation. If one assumes that only negative control elements obtain and that inactivation of regulatory elements does not occur, one can calculate that the regulatory components for transport are present in not more than a 30% excess in steady-state growthwith leucine, since a derepression in transport had occurred after a 30% increase in cell mass. Our tests for degradation of transport components indicated that inactivation was not a major factor in the regulation of transport. It appears that the decrease in uptake capacity of cells after addition of leucine is due to a greatly reduced differential rate of synthesis of these proteins relative to total cellular protein. In a study of the regulation of proteolysis in E. coli, Pine (40) noted that leucine was less effective as a proteolytic regulator than were other amino acids, such as phenylalanine. Our findings here are consistent with those considerations. Role of leucyl-tRNA and its synthetase in transport regulation. We have reported previously that the growth of an E. coli B/r mutant with a temperature-sensitive leucyl-tRNA synthetase at nonpermissive temperatures leads to derepression of transport and the leucine-binding protein (42). We have confirmed this earlier report by using an E. coli K-12 strain and have shown that neither the isoleucyl- nor the valyltRNA synthetase is involved in the regulation of transport. The derepression of transport in the leucyl-tRNA synthetase mutant in the presence of excess exogenous leucine indicates that leucine itself is not sufficient to generate a repression signal, but that it must interact with

J. BACTERIOL.

tRNA and a functional tRNA synthetase. Regulation of transport and biosynthesis. An examination of the quantitative aspects of transport regulation reveals some interesting features. A prototrophic strain (EO300) transports leucine with a specific activity of 0.66 U per g of cell (dry weight) when grown on MOPSG. The specific activity when grown with 0.4 mM leucine is 0.15 U/g (dry weight). When strain CU367 is limited for leucine, its derepressed transport level is very close to that of the prototrophic strain in minimal medium, 0.63 U/g. These considerations indicate that a prototrophic strain in minimal medium has a fully derepressed transport capacity for leucine, isoleucine, and valine. This is in striking contrast to the level of the leucine, isoleucine, and valine biosynthetic enzymes in prototrophic strains grown in minimal medium, which are 60 to 80% repressed (55). Ifthis relationship can be generalized, it may indicate a hierarchy of regulation of transport and biosynthesis of the branched-chain amino acids in E. coli. One could imagine that this difference in sensitivity to leucine limitation of transport and biosynthesis would allow the cells to respond with maximum efficiency to a wider range of culture conditions, as has been proposed to justify the multiple regulatory systems in histidine (48) and tryptophan biosynthesis (3). ACKNOWLEDGMENTS We wish to thank J. J. Anderson and H. J. Whitfield of The University of Michigan and H. E. Umbarger of Purdue University for generously supplying bacterial strains. This investigation was supported by Public Health Service grant GM11024 to D.L.O. from the National Institute of General Medical Sciences.

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AMINO ACID) TRANSPORT REGULATION

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