INTRODUCTION

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The aromatic amino acids phenylalanine, tyrosine, and tryptophan are actively transported across the inner membrane of the bacterium Escherichia coli by a number of distinct transport systems. A general aromatic transport system, encoded by the gene aroP, transports all three aromatic amino acids (1, 4). A closely related protein, PheP, is a high-affinity transporter of phenylalanine (23, 35). The PheP protein does not transport tryptophan and initially was not reported to be an active transporter of tyrosine. Tryptophan when present at a 20-fold excess can significantly inhibit phenylalanine transport by the AroP system but has only a slight effect on phenylalanine transport by the PheP protein. In the wild-type cell, expression of aroP is subject to control involving the TyrR protein (36), whereas the expression of pheP appears to be constitutive (23). The PheP protein is expressed to about one-third or less of the level of the AroP protein in derepressed strains. This weak expression can for the most part be attributed to the translational start codon GTG found in pheP. The AroP and PheP proteins are part of the amino acid transporter family within a ubiquitous superfamily of transporters referred to as the amino acid-polyamine-organocation superfamily (25, 38). Studies with uncouplers of oxidative phosphorylation and with strains deficient in F₀F₁-ATPase indicate that transport via the AroP and PheP systems is driven by the proton motive force (34).

The aroP and pheP genes show a high degree of sequence identity, encoding proteins composed of 457 and 458 amino acid residues, respectively (12, 23). In the predicted primary structure of the two proteins, 61% of the amino acid residues are identical. Secondary structure models for the orientation of the PheP and AroP proteins in the cytoplasmic membrane have been proposed based on their hydrophobicity profiles, distribution of charged amino acid residues, and a study of alkaline phosphatase sandwich fusions (6, 22). The topological models for the AroP and PheP permeases are very similar and involve hydrophobic nonpolar residues arranged in 12 transmembrane-spanning regions connected by hydrophilic segments. The two permeases show the greatest level of identity in transmembrane spans 1 and 2 and are least conserved in the amino and carboxyl termini and in transmembrane spans 11 and 12 (Fig. 1).

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Topological models of AroP (a) and PheP (b) permeases. The amino acid residues which are identical between AroP and PheP are shaded.

Despite the high degree of similarity between the AroP and PheP permeases referred to above, their substrate specificities and affinities differ. To investigate whether there might be distinct domains within their common structural arrangements which could be identified as being responsible for these differences, a number of AroP-PheP chimeric proteins were constructed and studied for their transport characteristics. In other studies, the characterization of chimeric proteins has proved very useful in investigating the structure-function relationship of a number of transporters (5, 9, 10, 11, 17, 30). A simple and efficient method for the construction of a series of AroP-PheP chimeric proteins via in vivo homologous recombination of their genes was suggested from the previous work of Tommassen et al. (30). In this study, we report the creation and the characteristics of a number of chimeric proteins involving both AroP and PheP and identify a residue present in AroP but not in PheP that is required for tryptophan transport. We also demonstrate that the inactivity of some chimeras is not a consequence of failure to be inserted into the membrane.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains, plasmids, and phages. The E. coli K-12 strains, plasmids, and phages used in this study are described in Table 1.

Growth media and reagents. The minimal media used were the half-strength buffer 56 of Monod et al. (16) and the 121 salts medium of Torriani (31), both supplemented with 0.2% glucose and the required growth factors. Kanamycin was added to nutrient and minimal media at a final concentration of 25 µg/ml. All enzymes were purchased from AMRAD Pharmacia Biotech, Melbourne, Australia, unless otherwise indicated. [-³⁵S]dATP (1,200 Ci/mmol; 10 mCi/ml) for use in DNA sequencing and L-[¹⁴C]tyrosine (493 mCi/mmol; 50 µCi/ml), L-[¹⁴C]phenylalanine (493 mCi/mmol; 50 µCi/ml), and L-[¹⁴C]tryptophan (52 Ci/mmol; 20 µCi/ml) for use in transport assays were obtained from NEN/DuPont. The chromogenic substrate 5-bromo-4-chloro-3-indolylphosphate-toluidine salt (XP) from Sigma Chemical Co. was dissolved in dimethyl formamide and used at 40 µg/ml in solid medium. ProtoGel 30% (wt/vol) acrylamide for protein gels was purchased from National Diagnostics. Oligonucleotides were synthesized on a Gene Assembler Plus (Pharmacia-LKB) or obtained commercially from Bresatec.

Recombinant DNA techniques. Standard recombinant DNA techniques were used essentially as described by Sambrook et al. (26).

Site-directed mutagenesis. The method of Vandeyar et al. (32) was carried out using a commercially available kit from United States Biochemical Corporation.

Construction of aroP-pheP chimeric genes. The strategy adopted for creating AroP-PheP chimeric proteins is summarized in Fig. 2. Plasmid pMU3322 used for the construction of aroP-pheP chimeric genes contains the aroP and pheP genes in tandem. This plasmid is a derivative of the low-copy-number vector pLG339 (copy number, ~6) (29). The vector M13tg130 (aroP) was digested with SalI and XbaI and cloned into the same sites of pMU2149 carrying the pheP⁺ gene. The resulting plasmid pMU3322 contains the aroP gene located upstream of the pheP gene, with unique XbaI and EcoRV sites located on the vector between the two genes. The plasmid was digested with both of these enzymes, the linear DNA was transformed into strain JP7910, and then kanamycin-resistant colonies were selected. The amount of linear DNA required to produce between 10 and 100 kanamycin-resistant transformants was determined to be between 0.5 and 1.0 µg per 200 µl of competent cells. The percentage of transformants containing chimeric genes ranged from 30 to 75% between individual experiments. A total of 11 transformants were characterized which contained plasmids harboring chimeric genes. To allow nucleotide sequencing of the chimeric genes, the 2.2-kb SalI-EcoRI fragment of pMU3322 derivatives harboring aroP-pheP chimeric genes was cloned into the same sites of M13mp18, and sequencing was performed using the dideoxy method of Sanger et al. (27).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Construction of aroP-pheP chimeric genes by in vivo homologous recombination. The homologous genes aroP (black arrows) and pheP (gray arrows) are located in tandem on plasmid pMU3322. Situated between these genes are unique XbaI and EcoRV restriction enzyme sites. The plasmid is cleaved with these restriction enzymes, and the linear DNA is transformed into the E. coli strain JP7910. The linear plasmid DNA cannot be maintained in the host, but stable circular plasmids can arise by a recombination event between homologous regions in the two genes.

Construction of aroP-pheP-aroP chimeric genes. Additional chimeric proteins between AroP and PheP were constructed by exchanging different regions of the chimeric genes generated by in vivo homologous recombination. A pheP-aroP chimeric gene (P365A) in which the site of recombination between the pheP and aroP genes occurred at nucleotide position 1110 was isolated by in vivo homologous recombination using a starting plasmid in which the order of the aroP and pheP genes was reversed to that of pMU3322. This pheP-aroP chimera was used to generate aroP-pheP-aroP chimeric genes. A unique SphI site is present at nucleotides 951 to 955 in the pheP gene (Fig. 3). Digestion of this pheP-aroP chimera with SphI and EcoRI (located downstream of the chimeric gene coding sequence) enables this 800-bp fragment to be cloned into the equivalent sites of the aroP-pheP chimeric genes, thus generating aroP-pheP-aroP chimeras. Three such AroP-PheP-AroP chimeras were constructed between chimera P365A and chimeras A80P, A152P, and A286P.

View larger version (85K): [in this window] [in a new window]   FIG. 3.   Sites of recombination of the aroP-pheP chimeric genes. The nucleotide sequences of the aroP (upper sequence) and pheP (lower sequence) genes are shown. Homologous nucleotide sequences are indicated by two dots. The sequences determined to be the site of genetic recombination for individual chimeric genes are boxed, with the corresponding chimeric gene numbers shown.

Chimeric AroP-PheP-alkaline phosphatase fusions. Plasmid pMU3767, which contains a pheP-alkaline phosphatase sandwich fusion at position aspartate 432, was digested with SphI and BamHI restriction enzymes. The 2.5-kb SphI-BamHI partial fragment containing the pheP-phoA fusion was cloned into the equivalent sites of pMU3322-derived aroP-pheP chimeras A80P, A152P, and A286P. The resulting aroP-pheP-phoA fusions contain alkaline phosphatase inserted at position aspartate 432, which is a proposed periplasmic location. Additional alkaline phosphatase fusions were constructed with chimera A286P. In this case, oligonucleotide-mediated site-directed mutagenesis (32) was used to create individual unique BglII restriction sites within the chimeric A286P gene present on M13mp18. The 'phoA gene from pSWFII (8) was excised on a BamHI fragment and inserted in frame into the chimeric gene at each of the BglII sites. The detailed procedure for the generation of site-specific alkaline phosphatase sandwich fusions has been previously reported (6). Alkaline phosphatase sandwich fusions were constructed at positions isoleucine 169, alanine 224, proline 261, leucine 279, and leucine 361 in chimera A286P. An alkaline phosphatase sandwich fusion was also generated by this method at position leucine 279 in chimera A80P.

Transport assays. Cultures of JP7910 harboring the chimeric genes were grown in half-strength buffer 56 containing 0.2% glucose, the appropriate growth factors, and kanamycin at 37°C to an optical density at 600 nm of 0.45 to 0.55. Transport activity was assayed as previously described (37) in the presence of 10 µM L-[¹⁴C]tyrosine, L-[¹⁴C]phenylalanine, or L-[¹⁴C]tryptophan. Competition studies were performed by measuring transport activity in the presence of a 20-fold excess of unlabeled amino acid.

Alkaline phosphatase assays. Cultures of JP8442 harboring aroP-pheP-phoA gene fusions were grown under the same conditions described for the transport assay, and alkaline phosphatase activity was assayed as described by Manoil (14). Each assay was performed in duplicate on at least three separate occasions.

Immunoprecipitation. Preparation of cell extracts and immunoprecipitation conditions were performed and set, respectively, according to the method specific for integral membrane proteins described by Ito and Akiyama (13), using anti-PheP serum TTP7 (21). Samples were electrophoresed on a sodium dodecyl sulfate-12% polyacrylamide gel. The dried gel was exposed to X-ray film for at least 72 h. Broad-range protein molecular weight standards (Bio-Rad) were used to estimate the molecular weights of PheP and chimeric proteins. The densities of the radioactive bands were measured by scanning the autoradiographs with a Molecular Dynamics scanning densitometer, and these values were used to estimate the levels of chimeric proteins relative to that of the wild type.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Localization of the sites of recombination in the chimeric genes. A range of AroP-PheP chimeras were produced as described in Materials and Methods, and the fusion sites in the 11 chimeric genes chosen for study were mapped using restriction enzymes that cleave in either aroP (DdeI, PvuII, AccI, and NdeI) or pheP (HpaI, BssHII, and AatII), but not in both. By determining the presence or absence of these cleavage sites in each chimeric gene, it was possible to localize the approximate sites of recombination of the individual chimeric genes. The exact sites at which recombination between the two permease genes had occurred were subsequently identified by nucleotide sequencing of each of the chimeric genes. The results are shown in Fig. 3.

Altered properties of the PheP system when expressed from aroP_P. Before describing the transport activities of the various chimeric proteins, it is necessary to describe an unexpected observation involving strains in which the pheP gene is transcribed from the aroP promoter and the pheP message is translated using the aroP translation initiation region involving an ATG instead of a GTG initiation codon. Since this will be the situation with each of the AroP-PheP chimeras, we made this strain as an appropriate control along with wild-type aroP for comparing transport activities.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Uptake of phenylalanine (), tyrosine (), and tryptophan () (10 µM) by E. coli strain JP7910 carrying pMU4784 with pheP expressed from the aroP transcription and translation regions (a) and pMU2195 carrying aroP (b). DW, dry weight.

Nomenclature. In order to simplify the description of the various chimeras, we have used the AroP residue number at each of the fusion sites. In the AroP-PheP chimeras, we have indicated after "A" the last AroP-specific residue in the chimera. In some cases, the actual recombination may have taken place a little further into the gene, but the additional AroP DNA sequence, although different from PheP, did not result in any changes at the amino acid level. In the case of the AroP-PheP-AroP chimeras, the fusion point for the distal AroP segment is indicated by the first AroP-specific residue after the PheP sequence.

Overall transport activity of the various chimeras. The overall transport activities of AroP, PheP, and each of the chimeric proteins are shown in Fig. 5, along with a diagram indicating the composition of the chimeras (the exact fusion point for each chimera can be established by consulting Fig. 1).

View larger version (36K): [in this window] [in a new window]   FIG. 5.   Schematic representation of the AroP permease (black), the PheP permease (grey), and each chimeric protein, illustrating the composition of AroP and PheP regions (black and grey). Based on topological information, the proteins are represented as possessing 12 membrane-spanning domains with their amino and carboxyl termini within the cytoplasm. The steady-state levels of phenylalanine, tyrosine, and tryptophan transport by derivatives of the transport-minus strain JP7910 carrying plasmids with these chimeric genes are also presented. Background levels of transport into the transport-minus strain have been subtracted. DW, dry weight.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   Uptake of phenylalanine (), tyrosine (), and tryptophan () (10 µM) by E. coli strain JP7910 carrying plasmids pMU6542 and pMU6543, encoding chimeric proteins A94P and A107P, respectively. DW, dry weight.

Identification of a residue critical for tryptophan transport. Reference to the AroP and PheP sequences in Fig. 1 reveals that the only differences between the two proteins in the region between positions 94 and 107 in AroP are leucine 102, tyrosine 103, and alanine 107, which in PheP are replaced by methionine, phenylalanine, and glycine, respectively.

Site-directed mutagenesis of PheP. To determine whether the substitution for phenylalanine 111 (equivalent to position 103 in AroP) of tyrosine was all that was required to enable PheP protein to transport tryptophan, site-directed mutagenesis was used to change phenylalanine 111 to tyrosine in the wild-type PheP protein. The pheP-carrying plasmid pMU3138 was the target for the mutagenesis, and the plasmid with the mutated pheP gene was introduced, as before, into the transport-negative strain JP7910. The transport properties of this new strain are shown in Fig. 7.

View larger version (13K): [in this window] [in a new window]   FIG. 7.   Uptake of phenylalanine (), tyrosine (), and tryptophan () (10 µM) by E. coli strain JP7910 carrying plasmid pMU3138 carrying wild-type pheP with an ATG start codon (a), pMU6557 carrying pheP with an ATG start codon and phenylalanine 111-to-tyrosine alteration (b), and pMU6558 carrying aroP with a tyrosine 103-to-phenylalanine alteration (c). DW, dry weight.

The significance of tyrosine 103 in AroP. To determine the role of tyrosine 103 in the AroP protein, site-directed mutagenesis was used to change tyrosine 103 to phenylalanine in the wild-type aroP gene carried on plasmid pMU2195. The mutated gene was again introduced into the transport-negative strain JP7910, and the new strain was tested for its ability to transport each of the three aromatic amino acids. The results are shown in Fig. 7. Although the tyrosine 103-to-phenylalanine alteration does not completely abolish tryptophan transport, it does specifically decrease it to less than 50% of wild-type levels, and it also reduces tryptophan inhibition of phenylalanine transport from 95 to 62%. From this result, it would appear that tyrosine 103 does play an important role in tryptophan transport but that in AroP protein it is supported by other AroP-specific residues beyond span III. It should also be noted that whereas tyrosine at a 20-fold excess can inhibit phenylalanine transport by more than 90% in wild-type AroP, in the case of the tyrosine 103-to-phenylalanine mutation this inhibition is reduced to only 50% (data not shown).

The failure of some chimeras to transport any amino acids. As previously stated, a number of chimeras fail to transport any amino acids, and in particular, whereas chimeras A152P and A80P368A fail to show any significant transport activity, chimeras A152P368A and A80P are two of the most active chimeric transporters. By using an anti-PheP specific polyclonal serum that had been prepared against residues 231 to 245 of PheP (see Materials and Methods), we carried out pulse-chase experiments to test whether (a) the nontransporters failed to insert into the membrane or (b) whether such insertions were inherently unstable. The results are shown in Fig. 8.

View larger version (21K): [in this window] [in a new window]   FIG. 8.   Stability of chimeric proteins. E. coli strain JP7910 expressing wild-type PheP, AroP-PheP, and AroP-PheP-AroP chimeric proteins was pulse-labeled with [35S]methionine-cysteine for 1 min followed by chase periods of 1, 5, 15, and 30 min. A 0.5-ml aliquot of each sample was taken at the times indicated and immunoprecipitated with the PheP-specific antibody TTP7. The samples were separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and visualized by autoradiography. The intensity of each band was quantified by densitometry as described in Materials and Methods. The arrow indicates the location of bands corresponding to wild-type PheP and AroP-PheP chimeric proteins.

Identification of residues affecting transport activity of different chimeras. We have used site-directed mutagenesis to investigate the particular amino acid substitutions responsible for major changes in the transport activities of the different chimeras, and the results are shown in Table 4. Chimera A80P has twice the transport activity of A57P and differs from it by the substitution of an alanine for serine at position 76 and a substitution of a serine for an alanine at position 80. When we separately changed these residues in A57P, the change of serine to alanine at position 76 resulted in a doubling of activity, whereas the change of alanine to serine at position 80 had no effect. Chimera A94P shows a specific decrease in the steady-state levels of tyrosine accumulation and differs from A80P at two positions. Single-site substitutions in A80P reveal that this change is a consequence of the substitution of serine for proline at position 89 and that the other change of leucine to alanine at position 94 has no effect (data not shown). As we have already described, the significant loss of activity in A107P can be attributed to the change of glycine to alanine at position 107. 

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of an amino acid of major importance for tryptophan transport. A system of in vivo homologous recombination has been successfully used to generate a family of AroP-PheP chimeras. Examination of these different chimeras coupled with some site-directed mutagenesis has identified an amino acid, phenylalanine 111 in span III of PheP, which, when changed to tyrosine (the corresponding residue in AroP), enables the protein to transport tryptophan and enables tryptophan to inhibit the transport of phenylalanine. The observation that cold tryptophan can inhibit the transport of both phenylalanine and tyrosine would suggest that the substrate binding sites for each of these amino acids overlap and certainly involve span III. We have not measured the K_m for tryptophan transport in this PheP mutant, but the observation that a 20-fold excess of cold tryptophan causes a 60% inhibition of phenylalanine transport rather than the >90% seen in AroP and in the chimera A107P indicates that other AroP-specific amino acids in A107P may also contribute to tryptophan binding or transport. The finding that the conversion of a phenylalanine to tyrosine is necessary for tryptophan transport implies some essential function for the hydroxyl of the tyrosine residue. The observation that it is possible, in computer modeling, to position either a tryptophan or a tyrosine molecule so that it is simultaneously hydrogen bonded to both glutamate 118 and tyrosine 111 may be relevant to the elaboration of a molecular mechanism for the transport of these amino acids, although it should be noted that similar hydrogen bonds can be formed with glutamate 118 and tyrosine 125 and yet changing tyrosine 125 to phenylalanine does not interfere with either tryptophan or tyrosine transport.

Reevaluation of span III. The composition of span III in both PheP and AroP has not been as clearly defined as that of most other spans. Hydropathy algorithms such as GES or von Heijne's Top Pred select a sequence from 98 to 116 in PheP and from 91 to 109 in AroP. However, a study of alkaline phosphatase sandwich fusions with both PheP and AroP produced results indicating that position 116 in PheP and 109 in AroP were located well within the membrane, and on the basis of these results, the sequences 106 to 126 and 98 to 118 have been selected for span III in PheP and AroP, respectively (Fig. 1) (6, 22). In the PheP sequence from 98 to 126, there are seven aromatic amino acids. If this is represented as a helix, all of these residues, phenylalanine 98, phenylalanine 101, tryptophan 105, tyrosine 107, tryptophan 108, phenylalanine 111, and tyrosine 125, are located on the same side of the helix, which also includes an essential and highly conserved residue, glutamate 118, and the two important residues glycine 115 and alanine 122 (Fig. 9).

View larger version (33K): [in this window] [in a new window]   FIG. 9.   A helical wheel plot of PheP amino acid residues 98 to 126, assuming a periodicity of 3.6 residues per turn. Aromatic amino acid residues (boxed) and residues found to be important for activity (underlined) cluster on one side of the helix.

The basis for loss of activity in some chimeras. An examination of chimeras which had lost all activity revealed that this was not a consequence of failure to be inserted into the membrane. A detailed study of one such chimera, A152P, revealed two amino acid substitutions, alanine for glycine 115 and valine for alanine 122, which contributed to loss of activity. Activity could be partially restored by reversing either of these substitutions. These residues are located above and below glutamate 118 slightly to one side (Fig. 9). Since alanine and valine at these positions are no encumbrance to activity in the case of AroP and since, when distal AroP spans are changed in A152P to produce A152P368A, activity is restored, we propose that important interactions occur between these residues and others located in distal spans of the protein. One consequence of this interaction could be the positioning of the negatively charged residue glutamate 118. At the position corresponding to 118 in PheP, other bacterial members of the family contain either glutamate or aspartate. Converting glutamate 118 of the PheP protein to glycine, valine, leucine, tryptophan, or asparagine has been shown to completely destroy transport activity, whereas changing it to aspartate reduces transport activity to 36% of wild-type level (24). These results indicate the importance of glutamate 118, which is presumed to have a critical role either in proton translocation or in substrate binding or both. The observation that its replacement by aspartate causes a partial reduction in activity suggests that span III has some flexibility to adjust to changes in side chain length. We have not yet identified the residue with which glutamate 118 interacts, but it should be noted that lysine 168 is also highly conserved and critical for transport activity. The replacement of lysine 168 of the PheP protein by arginine results in a reduction of transport activity to about 30% of wild-type level. It is possible that increasing the side chain length of glycine 115 and alanine 122 interferes with transport activity because it perturbs the positioning of glutamate 118 in the tertiary configuration of the PheP protein. Neither the glycine nor the alanine in span III at position 115 or 122, respectively, is highly conserved throughout other members of the family, and this may support the hypothesis that their significance is more related to packing arrangements with other helices than to providing, in the case of glycine 115, the sort of flexibility that has been recently demonstrated to be of importance in the Lac permease (33).