Leucine binding protein and regulation of transport in E. coli

zyxwvu zy Journal of Supramolecular Structure 6:419-431 (1977) Molecular Aspects of Membrane Transport 28 1-293 zy zyxwvut Leucine Binding Protein and Regulation of Transport in E. coli Dale L. Oxender, James J. Anderson, Mary M. Mayo, and Steven C. Quay zyx zyxwvutsr Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48 709 Leucine is transported into E. coli cells by high-affinity transport systems (LIV-I and leucine-specific systems) which are sensitive to osmotic shock and require periplasmic binding proteins. In addition leucine is transported by a low-affinity system ( LIV-11) which is membrane bound and retained in membrane vesicle preparations. The LIV-I system serves for threonine and alanine in addition to the 3 branched-chain amino acids. The LIV-I1 system is more specific for leucine, isoleucine, and valine while the high-affinity leucine-specific system has the greatest specificity. A regulatory locus, livR at minute 2 2 on the E. coli chromosome produces negatively regulated leucine transport and synthesis of the binding proteins. Valineresistant strains have been selected t o screen for transport mutants. High-affinity leucine transport mutants that have been identified include a LIV-binding protein mutant, livJ, a leucine-specific binding protein mutant livK and a nonbinding protein component of the LIV-I system, l i v l f . A fourth mutant, /ivP, appears to be required only for the low-affinity LIV-I1 system. The existence of this latter mutant indicates that LIV-I and LIV-11 are parallel transport systems. The 4 mutations concerned with high-affinity leucine transport form a closely linked cluster of genes on the E. coli chromosome at minute 74. The results of recent studies on the regulation of the high-affinity transport systems suggests that an attenuator site may be operative in its regulation. This complex regulation appears t o require a modified leucyl-tRNA along with the transcription termination factor rho. Regulation of leucine transport is also defective in relaxed strains. Among t he branched-chain amino acids only leucine produces regulatory changes in LIV-I activity suggesting a special role of this amino acid in the physiology of E. coli. It was shown that the rapid exchange of external leucine for intracellular isoleucine via the LIV-I system could create a n isoleucine pseudoauxotrophy and account for the leucine sensitivity of E. coli. Key words: regulation, amino acid transport, mutants, leucine sensitivity, leucine, isoleucine, valine E. coli have developed 2 basic types of active transport systems for leucine. One of these transport systems is a low-affinity membrane-bound system (LIV-11) and can be observed in membrane vesicle preparations described by Kaback (1). High-affinity trans- Received May 27, 1977; accepted June 3, 1977 zyxwvu 0 1977 Alan R . Liss, Inc., 150 Fifth Avenue, New York, NY 1001 1 42O:JSS zyxwvutsrq Oxender et al. port systems for leucine (LIV-I and leucine-specific) in E. coli are sensitive t o osmotic shock (2) and require periplasmic binding proteins. The leucine binding proteins have been described in various review articles (3-5). The high-affinity LIV-I system has a K, for leucine o f lop7 M and also transports isoleucine, valine, threonine, and alanine. The leucine-specific system comprises about 20% of the high-affinity transport capacity for leucine of wild-type E. coli K-12. The regulation of the high-affinity transport systems which respond to the level of leucine in the medium has been extensively studied in our laboratory (6-10). Mutations resulting in a loss of leucine repression have been mapped and characterized (6, 10). The mutations livR (signifying a derepression of the LIV-I and leucine-specific systems) and IstR (signifying derepression of the leucine-specific system and the LIV-I1 systems) determine negatively acting regulatory elements and are genetically closely linked. Our current understanding of the regulation of leucine transport suggests that leucine interacts with t RNAleu and the leucyl-tFWA synthetase t o produce this regulation. The regulation primarily changes the differential rate of synthesis o f transport components relative to total cellular proteins (9). We have recently shown that mutations that alter the hisT gene which codes for a tRNA modifying enzyme (1 1) and the rho allele which codes for a transcription termination factor (1 2) also produce a derepression of leucine transport (1 3, 14). These results suggested that an "attenuator" type of regulation obtains similar to that described for the regulation of tryptophan (15), histidine (16), and branchedchain amino acid (1 1) biosynthetic pathway enzymes. Analysis of the structural components of the high-affinity and low-affinity transport systems for leucine has been aided by our recent identification (17) of a genetic complex which codes for at least 4 functions involved in binding protein expression and LIV-I1 transport expression. This complex is linked t o malT o n the E. coli genetic map, approximately 180" from the regulatory loci livR and IstR. zyxwvu zyxw zy METHODS Growth Conditions Growth supplements for auxotrophic strains when not indicated otherwise were: 5 0 pg/ml for all L-amino acids except for L-leucine which was used at a concentration of 25 pg/ml; thymine 50 pg/ml; vitamins 1 pg/ml, glucose 0.2%. For all experiments the medium consisted of a morpholino propane sulfonatebuffered salts solution (MOPS) described by Neidhardt et al. (1 8). All supplements were either sterilized by filtration through 0.2 ym membrane filters or autoclaved. Cultures were grown aerobically in 125- or 250-ml erlenmeyer flasks in a shaking water bath (New Brunswick Scientific Company, Model G-76) that maintained constant temperatures between 30 and 41 ? 0.25"C. The platform rotation was approximately 150 rpm. Cell growth was followed b y monitoring absorbance at 420 nm at room temperature using a GCA-McPherson model EU 707-1 2 spectrophotometer. Isolation of Binding Protein Binding protein was isolated by the osmotic shock procedure and binding activity determined by equilibrium dialysis as described previously (5). Transport Assays Routine transport assays, as well as the rapid transport assay variant for screening large number o f prospective transport mutants, were performed as described previously (10). 282:MAMT zyxwvu zy zyx zyxwvut Leucine Transport and Regulation in E. coli Enzymes Assays JSS:421 The following enzymes were assayed as referenced: Threonine deaminase: (E. C. 4.2.1.6; L-threonine hydrolyase [deaminating] : (threonine dehydratase) (19)except that the absorbance of the 2,4-dinitrophenylhydrazone derivative of a-ketobutyrate was measured at 530 n m in a Zeiss PMQ2 spectrophotometer. Acetohydroxy acid synthetase: (AHAS; acetolactate synthetase, E.C.4.1.3.18) (20) 3-isopropylmalate dehydrogenase (IPMP; 2-hydroxy-4-methyl-3-carboxyvaleratenicotinamide adenine dinucleotide oxidoreductase. E. C. 1.1.1.85)(21). Mu Phage Procedure Wild type Mu-1and Mucrs 62 lysates were prepared as already described (17). Mu Mutagenesis E. coli cultures were grown to a density of 2 X lo8 cells/ml in LBT broth supplemented with 0.01M MgS04 and mutagenized b y adding Mu or M u m phage to a multiplicity of infection of 1 .O. After lysis at the appropriate temperature (37°C for Mu, 30°C for M u m ) the cultures were allowed to grow overnight. Procedures for detecting Mu lysogeny have been previously described (1 7). SDS-Polyacrylamide Gel Electrophoresis Slab gels containing 1 1% acrylamide and 0.1%SDS (sodium dodecyl sulfate) were prepared by the method of Laemmli (22). The gels were 1.5 mm thick. Samples of concentrated shock fluids were prepared in 1% SDS by heating for 2 min at 100°C.After electrophoresis the gels were fixed, stained, and destained by the method of Fairbanks (23). The gels were dried on filter paper under vacuum and photographed. RESULTS zyxwvutsrq Isolation of Mutants in the Branched-ChainAmino Acid Uptake Systems Regulatory mutants. Among spontaneous mutants selected for the ability t o grow on D-leucine were mutants with elevated levels of branched-chain amino acid transport (6). The mutant allele livR results in failure of L-leucine to repress the osmotic shock-sensitive high-affinity LIV-I and leucine-specific transport systems and their respective binding proteins, but has no apparent effect on the membrane-bound low-affinity LIV-I1 system (6, 10). The lsrR allele, However, results primarily in a derepression of the leucine-specific transport system and in the level of leucine-specific binding protein, with an apparent twofold increase in the LIV-I1 system as well (10 and unpublished experiments). The IstR mutation permits the cells t o utilize lower levels of D-leucine than the livR mutation (Table I). Since these 2 distinct patterns of regulation suggested that different functions were specified by the livR and lstR alleles, a complementation test was carried out between lsrR and livR mutants. Strains were constructed which were diploid for the E. coli chromosome covering the region in which lsrR and livR are located (Fig. 1). Previous work had indicated very close linkage between these 2 alleles (10). Both lsrR and livR are recessive t o their respective wild-type alleles (Table 11, strains 1 , 2 , 3 , and 5) which therefore indicates that they produce a negatively operating, diffusable factor. The merodiploid strain listed as number 4 in Table I1 is homozygous for IstR and displays the mutant phenotype. Strains homozygous for livR also display the mutant phenotype (10).Strain number 6 , however, shows that the wild-type alleles of livR and lstR can complement MAMT:283 422:JSS zyxwvutsrq zyxwvut O x e n d e r e t al. zy zyxwvutsrq TABLE I. Growth Phenotypes of liv Mutants Colonv formationd o n medium suoolemented with 0.03 uM L-leucineb Mutationa liv+ livR lstR livR livJ 1ivR livK livR livH lstR livH livP 0.4 p M L-valinec 850 pM D-leucineb 1,700 pM D-leucineb t + t t t t - aComplete strain descriptions are published elsewhere; all strains are isogenic and carry the derepressed transport allele livR (10) except IivH livP, which is in a lstR strain background. bDetermined in leu strain background CDetermined in leut strain background dDetermined after 72-h incubation at 32°C zyxwvutsrq zyxwv TABLE 11. Complementation Analysis of lstR and livR Alleles in F-prime Merogenotes Phenotype Merogenote genotypea 1. F > F 2. 3. 4. 5. 6. + t F + i t livR + F t + + + + lstR + t F +- + lstR lstR lstR lstR + F - livR + L-leucine uptake, nmol/min/mg dry wt. Colony formation, 1,700 pM D-leucine 0.020 - 0.012 - 0.020 - 0.052 t 0.03 - 0.018 - astrains carrying the F147 gal+ F-prime (35) were constructed from recipients which were, in addition to the designated transport alleles, recA gal leu. They were grown o n galactose minimal medium supplemented with 25 pg L-leucine per ml. The F147 istR episome was isolated from a D-leucine utilizing homogenoteof a recA+ strain similar in genotype to strain No. 3, above. each other, y i e l d i n g the wild-type phenotype. We conclude from these results t h a t ZivR and ZstR are separate genes, each c o n t r o l l i n g a d i f f e r e n t pattern of regulation of the branched-chain amino acid uptake systems. The m o l e c u l a r n a t u r e of these g e n e p r o d u c t s is under investigation. 284:MAMT zyxw zy zyxwvut Leucine Transport and Regulation in E. coli (liv K, J ) liv H P aed ma1 T JSS:423 I! aro B rpe L Fig. 1. Simplified genetic map of the E. coli chromosome taken from Bachpan, Low, and Taylor (24) showing positions of Ziv mutations discussed within. Alleles in parentheses have not been precisely mapped. zyxwvut zyxw Isolation of Mutants Defective in Branched-Chain Amino Acid uptake Systems Mutant strains possessing the livR allele have increased wnsitivity to L-valine, which inhibits the growth of E. coli K-12 (Table I). Mutations to Gvaline resistance Were induced by the mutator phage Mu in strains possessing the livR allele (Table I). A concentration of L-valine was chosen which would prevent colony formation only in strains with elevated levels of the LIV-I system in order to avoid excessive selective pressure (17). Three biochemically distinct classes of mutants were found which have been designated livH, livJ, and livK (17). These map together by transduction and are closely linked to malT (17 and Fig. 1). The transport of L-leucine in these strains has been diminished (Table 111) without affecting the transport of other amino acids (17). The mutation livK results in a loss of all detectable high-affinity leucine uptake (LIV-I and leucine-specific systems) and a loss of the leucine-specific binding protein, both by in vitro activity (Table I) and by SDSpolyacrylamide gel electrophoresis (Fig. 2). The LIV-binding protein is still present in these strains even though all high-affinity transport is lost. The high-affinity uptake of L-valine and L-isoleucine has also been eliminated (17), as has the ability of the cells to utilize D-leucine (Table 1). The livJ mutation eliminates the LIV-I high-affinity uptake but the leucine-specific system and LIV-I1 system are unaffected. There is a parallel loss of the LIV-binding protein activity and the corresponding band on SDS-polyacrylamide gel slabs (Table 111 and Fig. 2 ) . D-Leucine utilization, however, is retained (Table I) indicating that the D-leucine utilization phenotype in ZivR and lstR strains is presumably due to a derepression of the leucine-specific system. The mutation livH eliminates both high-affinity transport systems without producing any detectable change in the mobilities or activities (Table 111, Fig. 2 and Ref. 17) of the 2 binding proteins. Evidently livH codes for an additional factor of high-affinity uptake, at present unidentified. MAMT:285 zyxwvutsrq zyxwvuts zyxwvu zyxwvutsr zy 424:JSS Oxender e t al. TABLE 111. Transport Phenotypes of liv Mutants zyxwvu zyxwvuts zyxwvut Percent of wild-type Mutationa liv+ livK livJ livH livH livP leucine uptakeb 100 10 55 15 2 L-leucine-binding protein activityd L-leucine uptake systemsc LIV-I Leucine-specific LIV-I1 + + t t - - - t + - t - ~ LIV t + Leucine-specific + - - t + + + + aStrains, same as Table I bL-leucine transport was measured at 5 pM L-leucine. cThe leucine-specific and LIV-I systems are defined by their low Km (0.1-0.4 p M ) for L-leucine; the leucine-specific system resists inhibition by L-isoleucine and is responsible for D-leucine uptake. The LIV-I1 system has a higher K, (2-4pM) for L-leucine and also transports L-isoleucine and L-valine. Presence or absence of a given system was determined by kinetic analysis over substrate ranges from 0.02 p M to 20 p M ( 6 ) . dL-leucine-binding activity was measured on crude osmotic shock fluid; the leucine-specific binding protein activity was determined by resistance of L-leucine binding to L-isoleucine competition (10). A third group of mutants were obtained which were defective in the low-affinity (LIV-11) transport system. These mutants were obtained b y inducing a second mutation in the livH mutant strain b y mutagenizing the livH strain with ethyl methane sulfonate and penicillin-enriching for mutant strains unable t o utilize low concentrations of L-leucine (Table I). One of these mutations has been designated IivP. Conjugational analysis has shown that livP is also linked t o malT (unpublished observations, Fig. 1). Transport of L-leucine in the double mutant livH livP has been dramatically lowered over that of the parent livH strain (Table III), and kinetic analysis (data not shown) has revealed that the LIV-I1 system has been reduced t o undetectable levels. Thus, in this double mutant strain both high- and low-affinity transport systems for leucine have been mutationally eliminated. Other workers, however (25, 26) have reported additional low-affinity uptake systems for leucine. We are presently characterizing the remaining leucine uptake in the double mutant strain t o ascertain the nature of the residual leucine uptake. A genetic complex is suggested for leucine transport genes in E. coli by these studies since at least 4 different genes specifying components of the known transport systems for leucine map in the same region. We believe that the structural genes for the leucine-binding proteins are part of this complex since a previously reported mutation altering the structure of the leucine-specific binding protein (6) also maps in this area (17). We are presently characterizing other mutants by complementation analysis and fine mapping t o better define the genetic organization of the region. We are also attempting t o biochemically identify the components coded for b y the livH and livP genes. Regulation of Leucine Transport Repression by leucine. Early studies showed that high-affinity leucine transport activity is highly regulated and responds t o the level of leucine in the growth medium (6). There is a direct relationship between the level of high-affinity leucine transport and the 286:MAMT Leucine Transport and Regulation in E. coli LS JSS:425 zy zyxwv zyxwv -87 LIV -OP zy zyxwvutsrq I:ig. 2. Sodium dodecyl sulfate-polyacrylamide gel profile of crude osmotic shock fluid proteins. The slab gels were prepared according to the method of Laemmli (22) and were 11% acrylamide. Direction of migration is from top t o bottom. A sample of pure LIV-binding protein (LIV-BP) and a trace of the leucine-specific binding protein (LS-BP) are shown o n the left; H) shock fluid from a strain carrying the liuH mutation, J) strain with liuJ mutation, and K ) strain with liuH. The wild-type profile (not shown) is identical to H. level of the leucine-binding proteins (1 7). The correlation between the transport capacity and the amount of binding protein shows that the binding proteins are the rate limiting component in the transport of leucine into E. coli. When E. coli is grown on nutrient medium or minimal medium containing 25-50 pg/ml L-leucine essentially complete repression of the synthesis of both the LIV and the leucine-specific binding proteins occurs and essentially all high-affinity leucine transport is abolished (27). MAMT:287 426:JSS zyxwvut zyxwvutsrq Oxender e t al. Separate Regulation of Biosynthesis and Transport of Leucine The levels of the leucine biosynthetic enzymes are also regulated by the intracellular level of branched-chain amino acids (28). Since both transport and biosynthetic enzymes for leucine were repressed by growth on leucine we attempted to determine whether these 2 processes were regulated in a concerted manner. For these studies we collaborated with Dr. Umbarger of Purdue University. We provided Dr. Umbarger with mutant strains that were derepressed for leucine transport and he gave us several mutants that were derepressed for the biosynthetic enzymes of the branched-chain pathway. We examined the regulation of leucine transport in strains that were derepressed for biosynthesis (IeuABCD, ilvB, iIvADE) as well as a deletion of the entire leucine biosynthetic operon. Leucine transport activities and leucine-binding protein levels in these strains were regulated in a normal manner. Using the transport mutants from our laboratory, Dr. Umbarger showed that the regulation of the biosynthetic enzymes for the branched-chain amino acids was not altered in mutants with derepressed transport and leucine-binding proteins. The normal regulation of transport in E. coli strains with deletions of the entire biosynthetic operon indicated that none of the gene products of this operon are required for regulation of transport. We concluded from these studies that the transport systems and the biosynthetic enzymes in E. coli are not regulated together by a cis-dominant type of mechanism and although both systems appear to have components in common it is possible to obtain separate regulatory mutations for each system (7, 13). zyx Role of Leucyl t R N A Synthetase in Transport Regulation A possible candidate for a common component in the regulation of both transport and biosynthesis of leucine is the requirement for aminoacyl tRNA rather than the free leucine. Extensive studies in other laboratories have indicated aminoacyl-tRNA synthetases are part of the regulatory system for branched-chain amino acid biosynthesis (29). The availability of temperature-sensitive mutants for the leucyl-tRNA synthetase (leuSI) permitted us to determine whether this enzyme was also involved in the regulation of the transport of leucine. We examined both the level of transport activity and the level of the periplasmic-binding proteins in the temperature-sensitive mutant strain (leuS1) grown at the permissive temperature (36°C) and the nonpermissive temperature (41 "C). Strain EB143 has a complete deletion of the leucine biosynthetic operon which avoids the increased endogenous levels of leucine that would be produced in a prototrophic temperature sensitive IeuS strain grown at 41°C. Strain EB144 served as the isogenic non-temperaturesensitive control strain. The results are presented in Table IV. When mutant strain EB143 was shifted from 36 to 41°C the transport activity for leucine, isoleucine, and valine was greatly increased. Little effect was observed for proline or histidine uptake. Proline is transported by a membrane-bound transport system that derives its energy from the membrane potential while histidine is transported by a periplasmic-binding protein system and derives its energy more directly from ATP. The derepression of threonine deaminase that was observed for strain EB143 when it was shifted to 41°C was the expected response of a branched-chain amino acid biosynthetic enzyme which is subject to multivalent repression. The branched-chain amino acid transport system responds only to changes in the leucine level. Isoleucine and valine t RNA synthetase mutants did not produce regulatory changes in the transport activity. The shock fluid of strains EB143 and EB144 were examined for leucine-binding proteins when the cells were grown at 41°C. There is a fivefold derepression in the synthesis of the binding proteins for leucine when the temperature is shifted to 41°C 288:MAMT zyxw zyxwvutsrqpo zyxwvutsrq zyxwvutsr zyxwvutsrq zyxwvut L e u c i n e T r a n s p o r t a n d R e g u l a t i o n i n E. c o l i JSS:427 TABLE 1V. Expression of Transport and a Biosynthetic Enzyme Activity in Strains EB143 and EB144 Strain Growth Transport activitiesb Threonine conditionsa Leu I le Val dcaminasec EB 143, ura-leua1 101, IeuSl EB144, ara-leii~l101 E,0 143, ura-leii~llO1leu S l EB 144, aru-lennl I01 36°C 36°C 41°C 41°C 100 87 470 96 65 61 773 70 87 70 904 74 26 16 450 41 G r o w t h in glucose-basal salts medium plus 0.2 mM L-leucine. bTransport was assayed a t 1 pM leucine (Leu) or isoleucine (He) and 3 p M valine (Val). One hundred percent represents 0.23 mmol leucine taken up per min per kg cells dry weight. CSpecific activity represents pmol of a-ketobutyrate formed per min/g of cellular protein. The growth media included 0.4 mM L-leucine and L-isoleucine and 1 mM L-valine. zyxw zyxwvutsr with little change in the control strain. These results were consistent with a role of the binding proteins in the rate limiting step in leucine transport and further indicated that the synthesis of the binding proteins is regulated by the level of modified leucyl-tRNA or the leucyl-tRNA synthetase. We obtained a mutant strain with a defect in the maturation of leucyl-tRNA to distinguish between these 2 alternatives. Role of hisT Gene Product The hisT locus codes for an enzyme that converts uridine to pseudouridine in the tRNAs for leucine, histidine, and tyrosine. The regulation of biosynthetic enzymes for these 3 amino acids is no longer sensitive t o their cognate amino acids in strains containing a kisT mutation (1 1). We obtained E. coli strains containing a hisT mutation from Drs. R. P. Lawther and W. Hatfield. An examination of leucine transport activity in the hisT strain showed that the regulation was abnormal.The hisT strain showed a higher transport of leucine than the parental strain when grown under repressing conditions and furthermore it failed t o derepress leucine transport activity when grown under conditions of leucine limitation. Under these same conditions the biosynthetic pathway for leucine also does not show a repression or derepression regulation (Quay SC, unpublished observations). We reported similar results for a role of the hisT gene product in the regulation of leucine transport activity in Salmonella typhimurium (13). The results of these 2 studies with hisT strains provide evidence that the repression of transport of leucine requires fully maturated t RNA which is aminoacylated with leucine. Role of rho in Transport Regulation Recent reports have shown that several of the biosynthetic operons in bacteria contain an “attenuator” site near the operator region that acts as a barrier t o transcription by RNA polymerase (1 5, 16). The termination factor rho has been implicated in attenuator function. To test if transport for leucine, isoleucine, and valine is under an attenuator-type regulation 2 strains were obtained from Dr. Umbarger of Purdue University, which were leucine auxotrophs with one having a suA120 allele (1 4). The mAl2O allele is a missense mutation in the rho factor ( 3 3 ) . The initial rate of transport of 8 amino acids was measured in MAMT:289 428:JSS zyxwvutsrq zyxwvu Oxender e t al. TABLE V. Effect of a Mutation in Termination Factor rho o n Amino Acid Transport zyxwvutsrqp zyxwvut Specific transport activity (prnol/min/g cells, dry wt.) Control strain CU300a Substrate Arginine (3 pM) Glutarnine (1 pM) Histidine (1 pM) Isoleucine ( 1 pM) Leucine (1 p M ) Proline (3 pM) Tryptophan ( 3 p M ) Valine ( 3 pM) 2.92 1.90 0.20 0.12 0.10 0.17 0.5 1 0.31 SuA mutant CU2054a 3.61 0.63 0.38 0.24 0.19 0.17 0.55 0.61 "Cells were grown in MOPS-G, 0.2 m M leucine, and 25 rng/liter tryptophan. Cells were harvested and transport assayed as described previously (9). the rho mutant and the isogenic parental strain. The data are presented in Table V. The uptake of leucine, isoleucine, and valine is increased twofold even though leucine is present in the growth medium. Arginine and histidine transport activity is also increased in the rho mutant. Tryptophan and proline uptake is unchanged but glutamine is greatly decreased. The kinetics of uptake showed that the K, values for both the high- and the lowaffinity transport systems for leucine in the rho mutant were similar to those obtained for value for the high-affinity leucine uptake was increased the parental strain. The V,, approximately eightfold in the rho mutant. The V,,,,of the low-affinity system was only slightly elevated. An examination of the leucine-binding activity in the osmotic shock fluid was carried out. The leucine-binding activity showed a fourfold increase in the rho mutant. In a similar manner the histidine- and arginine-binding activities in the osmotic shock fluid were increased somewhat while glutamine-binding activity decreased significantly. These results show that rho-dependent transcriptional termination is important for leucinespecific repression of branched-chain amino acid transport (14). zyxw zyxwvutsrq Leucine Sensitivity The ZivR locus, which leads t o a trans-recessive derepression of the high-affinity transport system for leucine is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, serine, and certain analogues such as 4-azaleucine or 5',5',5'-trifluoroleucine (31). We recently showed that the ability of the LIV-I transport system to carry out exchange of endogenous amino acids for extracellular leucine is a major factor in leucine sensitivity. When E. coli cells are shifted from nutrient medium to minimal medium containing leucine a long lag time in the resumption of growth is observed. The lag time in the growth represents the time required to derepress certain biosynthetic enzymes. Added isoleucine antagonizes the leucine sensitivity showing that the necessary derepression of the biosynthesis of isoleucine is prevented by leucine. We were able to show that the necessary biosynthetic enzymes for isoleucine cannot by synthesized. The high-affinity transport system serves for rapid exchange of branched-chain amino acids while the low-affinity system does not produce significant exchange of these amino acids. 290:MAMT zyxw zyx zyxwvuts Leucine Transport and Regulation in E. coli JSS:429 These results provide a mechanism for leucine sensitivity and explain the increased sensitivity of strains with derepressed high-affinity transport such as that shown by the ZivR mutant strain. DISCUSSION The results presented above describe the 2 types of active transport systems in E. coli for the branched-chain amino acids. One type is membrane bound and can be observed in membrane vesicles (LIV-II), and the other type is osmotic-shock sensitive and requires binding proteins t o produce active transport of leucine (LIV-I). A regulatory genetic locus, livR, at minute 22 on the E. coli chromosome produces negatively regulated leucine transport and synthesis of the leucine-binding proteins. Valine-resistant strains that were still sensitive to the dipeptide glycylvaline were selected as potential LIV-I transport mutants. Among the valine-resistant mutants we identified a LIV-binding protein mutant (ZivJ), a leucine-specific binding protein mutant (livK), and a nonbinding protein component of the LIV-I system (livH). A fourth mutation, livP, appears t o be a component of the low-affinity LIV-I1 system. Since a livP mutant appears to have normal LIV-1 transport we have concluded that the LIV-I and LIV-11 systems represent parallel systems even though they may have certain components in common. These mutations concerned with leucine transport form a cluster closely linked t o rrzalT at minute 74 on the E. coli chromosome. The positions of these mutations are distinctly different from that of bmQ, brrzR, and brnS mutations of branched-chain amino acid transport described by laccarino and co-workers (26). Continued studies of the chemical and physical properties of the LIV- and leucinespecific binding proteins are being carried out in collaboration with other laboratories. Dr. Ovchinnikov and co-workers of Moscow, USSR, have recently published the complete amino acid sequence of the LIV-binding protein (32) and are currently sequencing the leucine-specific binding protein which is structurally very similar. Antonov et al. (33) have shown that a concentration-dependent reversible association of the LIV-binding protein may occur producing aggregates with an apparent molecular weight up t o 300,000. The presence of these aggregates has been correlated with nonlinear Scatchard plots of leucine-binding activity. Alternative explanations for nonlinear Scatchard plots of leucine binding t o the LIV-binding protein have been offered by Anraku and co-workers (34). These studies suggest that binding protein preparations contain bound ligand which could alter the specific activity of added ligand at high protein t o ligand ratios. Figure 3 presents a scheme of the regulatory components of the LIV-I transport system which responds t o the level of leucine added t o the medium. Modified leucyl-tRNA appears to play a role in the regulation along with the transcription termination factor rho. The results of this study suggest an attenuator site may be operative in the regulation of high-affinity leucine transport. This complex regulatory system for transport of leucine is similar t o that of its biosynthe.sis suggesting the important role of the LIV-I transport system to the physiology of the bacterial cell. Since changes in isoleucine or valine levels do not produce regulatory changes in LIV-I a special role of leucine in the physiology of E. coli is implied. The properties of the LIV-I system that give rise t o a rapid exchange of leucine for isoleucine, thus creating isoleucine pseudoauxotrophy under certain conditions, have helped t o explain the leucine sensitivity of E. coli (31). This complex regulatory system for the transport of leucine which is similar t o that of the biosynthetic enzymes provides an example of the important role transport systems can play in amino acid metabolism. MAMT:29 1 zyxwvuts zyxw zyxwvutsrqponmlkjihg Oxender et al. 43O:JSS I , Operator Structural Gene(s) I t RNA polymerase initiation I I rhomediated attenuation tRNA,,, + leucine leucyl-tRNA+ hisT t RNA Fig. 3. Model of leucine transport regulation in E. coli zyxwvutsrqp ACKNOWLEDGMENTS We wish t o thank Dr. H. E. Umbarger of Purdue University and Drs. R. P. Lawther and W. Hatfield of The University of California at Irvine for the E. coli strains containing the hisT mutation. This investigation was supported by Public Health Service Grant GMllO24 t o D.L.O. from the National Institutes of General Medical Sciences. REFERENCES zyxwv zyxw 1. Kaback HR: Methods Enzymol 22:99, 1971. 2. Neu HC, Heppel LA: J Biol Chem 240:3685, 1965. 3. Oxender DL: Annu Rev Biochem 41:777, 1972. 4. Oxender DL: In Manson LA (ed): “Biomembranes” New York: Plenum Press, 1975, vol5 p 25. 5. Oxender DL,Quay SC: In Korn ED (ed): “Methods of Membrane Biology.” New York: Plenum Press, 1976, vol 6, p 183. 6. 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