©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Conserved Motif, GXXX(D/E)(R/K)XGX(R/K)(R/K), in Hydrophilic Loop 2/3 of the Lactose Permease (*)

Amy E. Jessen-Marshall , Nanette J. Paul , Robert J. Brooker (§)

From the (1)Department of Genetics and Cell Biology and the Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul, Minnesota 55108

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine. Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high -turn propensity) may be structurally important at this position. The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity. In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low -turn propensity, still retained high levels of transport activity. Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues. However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose. Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this motif may be important in promoting global conformational changes within the permease.


INTRODUCTION

An important mechanism for the uptake of solutes by living cells involves membrane proteins known as cation/substrate cotransporters or symporters. These types of transport systems are widely found in many organisms including bacteria(1, 2) , fungi(3, 4) , plant(5, 6) , and animal cells(7, 8) . During uptake, symporters simultaneously couple the inwardly directed cation electrochemical gradient to the transport of solute so that secondary active transport can be achieved with regard to the solute(9, 10) . The lactose permease of Escherichia coli has provided a model system in which to investigate secondary active transport(11, 12) . This protein is found in the E. coli cytoplasmic membrane and couples the transport of H and lactose. From the cloning and nucleotide sequence of the lacY gene, the lactose permease contains 417 amino acids with a molecular weight of 46,504 (Refs. 13 and 14). Hydropathicity plots as well as genetic studies are consistent with a secondary structural model in which the protein contains 12 hydrophobic segments which traverse the membrane in an -helical manner(15, 16, 17) .

Recently, analyses of sequences among a variety of transporters has led to the conclusion that a large and diverse group of solute transporters are evolutionarily related to one another(18, 19, 20) . At the functional level, this superfamily includes proteins that transport a wide array of substrates such as sugars, Kreb's cycle intermediates, and antibiotics. For example, the lactose permease of E. coli, the glucose transporter of red blood cells, and bacterial tetracycline exporters are members. Mechanistically, this group is rather variable and includes proteins that catalyze uniport, symport, or antiport. This superfamily has been referred to as the USA()superfamily and as MFS (major facilitator superfamily)(20, 21) . Like the lactose permease, secondary structural models for members of the USA superfamily are commonly found to depict a membrane protein with 12 transmembrane segments traversing the membrane in an -helical manner. It has been proposed that this arrangement of 12 transmembrane segments may have arisen by a gene duplication/fusion event of an ancestral gene encoding a protein with six transmembrane segments(22) . More recently, we have proposed a three-dimensional model based on the structural characteristics of transmembrane domains, hydrophilic loops, putative helical interactions in the lactose permease, and rotational symmetry between the two halves of the protein(21) .

When comparing the sequences among distant relatives within the USA superfamily, it is frequently the case that relatively little overall homology is observed. Therefore, the superfamily has been divided into several groups (see Refs. 19 and 20). It is extraordinarily striking, however, that although members within different groups may bear little overall sequence homology, a motif, GXXXD(R/K)XGR(R/K), has been conserved throughout the entire superfamily(19) . Within secondary structural models, this peptide sequence is found in the hydrophilic loop between transmembrane segments 2 and 3, and repeated again between transmembrane segments 8 and 9. Although some variability is found within this motif, it has been fairly well conserved among all members of the superfamily. Indeed, it would appear that the presence of this sequence within loop 2/3 and 8/9 in a membrane protein is indicative of its appropriate placement within the superfamily. Upon careful inspection of both loop 2/3 and 8/9 among more members of the superfamily, it seems reasonable to suggest that the motif is D/E at the fifth position and R/K at the ninth position.

Only a few previous studies have involved perturbations in the structure within the loop 2/3 motif of the lactose permease. McKenna et al.(23) inserted two or six histidine residues between Leu-72 and Arg-73 within the loop 2/3 motif. These insertional mutants exhibited a marked defect in the downhill and uphill accumulation of lactose. Very recently, the role of glycine residues within the lactose permease has been investigated(24) . Even though none of the glycine residues within the permease were found to be essential for sugar transport, bulky substitutions at the glycine in the first position of loop 2/3 were found to be inhibitory. Finally, Wrubel et al. (25) examined the ability of portions of the lactose permease to be synthesized independently of each other and then reassemble into a functional permease. A mutant lacking amino acids 4 through 69 was coexpressed with a truncated form of the permease encoding amino acids 1 through 71. The results showed that these two pieces of the lactose permease were able to reassemble and produce a functional protein.

More detailed analyses of amino acids within loop 2/3 have been conducted on the tetracycline antiporter of E. coli (26-28). With regard to the conserved charged residues, it has been found that the negatively charged residue at the fifth position is critical for transport function. Of the three basic residues, only the one at the ninth position in the motif appeared to be important. The conserved glycines at the first and eighth positions were both found to be important for function with the first glycine being significantly more critical than the eighth. In the current study, a similar approach has been undertaken to examine the functional significance of the conserved amino acids within the loop 2/3 motif of the lactose permease.


MATERIALS AND METHODS

Reagents

Lactose (O--D-galactopyranosyl-[1,4]--D-glucopyranose) and melibiose (O--D-galactopyranosyl-[1,6]--D-glucopyranose) were purchased from Sigma. [C]Lactose was purchased from Amersham Corp. The remaining reagents were analytical grade.

Bacterial Strains and Methods

The bacterial plasmids are described in .

Plasmid DNA was isolated by the NaOH-sodium dodecyl sulfate method (29) and introduced into the appropriate bacterial strain by the CaCl transformation procedure of Mandel and Higa(30) .

Stock cultures of cells were grown in YT media (31) supplemented with tetracycline (0.01 mg/ml). For transport assays, cells were grown to midlog phase in YT media containing tetracycline (0.005 mg/ml) and 0.25 mM isopropylthiogalactoside to induce the synthesis of the lactose permease.

Sugar Transport Assays

For the experiments of I, midlog cells were washed in phosphate buffer, pH 7.0, containing 60 mM KHPO and 40 mM KHPO, and resuspended in the same buffer to a density of approximately 0.5 mg of protein/ml. Cells were then equilibrated at 37 °C and radioactive sugar (final concentration = 0.1 mM) was added. At appropriate time intervals, 0.2-ml aliquots were withdrawn and filtered over a membrane filter (pore size = 0.45 µm). The external medium was then washed away with 5-10 ml of phosphate buffer, pH 7.0, by rapid filtration. For the uphill transport experiments, 10 mM HgCl was included in the wash buffer to rapidly inhibit the lactose permease and thereby minimize sugar efflux during the removal of the extracellular medium. As a control, the lacY strain, HS4006/F`IZY/pACYC184, was also assayed for radiolabeled sugar uptake in order to obtain an accurate value for nonspecific sugar uptake.

HTransport

For the H transport experiments of Fig. 2, cells were grown to midlog phase and washed twice with 120 mM KCl. The cells were then suspended in 120 mM KCl and 30 mM potassium thiocyanate to a density of approximately 5 mg of protein/ml. 2.5 ml of cells were placed in a closed vessel with a lid containing tight-fitting openings for the insertion of a pH electrode, the introduction of nitrogen, and the insertion of (gas-impermeable) Hamilton syringes. Cells were made anaerobic under a continuous stream of nitrogen for at least 30 min. To initiate sugar-induced H transport, an anaerobic solution containing lactose was added to a final concentration of 10 mM. The change in external pH was measured with a Radiometer pH meter (PHM82) and electrode (GK2401C). Changes in pH were continuously recorded on a Radiometer chart recorder which had been modified to expand the scale of pH changes to a range where a 0.1-unit pH change caused a 10-cm deflection in the chart recording.


Figure 2: Sugar-induced H transport. H transport was measured in the strain T184 containing the designated plasmids as described under ``Materials and Methods.''



Site-directed Mutagenesis

The plasmid, pTE18 (Ref. 32) was digested with EcoRI to yield a 2300-base pair fragment containing the entire lacY gene. This fragment was ligated to the vector M13 mp18 (Ref. 33) in such a way that the antisense strand of the lacY gene was colinear with the plus-strand of the viral DNA. Site-directed mutagenesis was then performed by the method of Zoller and Smith (34) as modified by Kunkel et al. (35) using oligonucleotide primers producing the desired base change. Clones containing the appropriate mutation were identified by DNA sequencing (see below). The double-stranded replicative form DNA was then isolated and digested with EcoRI to produce the 2300-base pair fragment containing the lacY gene. This fragment was then ligated into the EcoRI site of pACYC184 (Ref. 36). Following transformation of E. coli strain T184, cells harboring hybrid plasmids with a lacY insert were identified by their loss of chloramphenicol resistance. The hybrid clones were restriction mapped to verify the orientation of the lacY gene on the plasmid. Only those clones which contained the lacY gene and the tetracycline resistance gene in the opposite transcriptional direction were used. The mutant plasmids were sequenced throughout the entire lacY coding sequence to verify the presence of the mutation and to be certain that no other secondary mutations had occurred. At least two independent clones for each mutant type were saved for further study.

DNA Sequencing

Single-stranded viral DNA was sequenced by the Sanger dideoxy method (37) using oligonucleotide primers which anneal within the lacY gene. Double-stranded plasmid DNA was isolated using Magic Minicolumns from Promega and sequenced according to Kraft et al.(38) .

Membrane Isolation and Immunoblot Analysis

For Western blot analysis, T184 cells containing the appropriate plasmid were grown as described above for the sugar transport assays. Ten ml of late log cells were pelleted by low speed centrifugation, quick frozen in liquid N, and thawed at room temperature. Cells were resuspended in 800 µl of MTPBS (150 mM NaCl, 16 mM, NaHPO, 4 mM NaHPO). Cells were quick frozen in liquid N and thawed twice. Cells were then sonicated three times for 20 s each. Membranes were harvested by centrifugation and resuspended in 100 µl of MTPBS. 100 µg of total membrane protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electroblotted to nitrocellulose and then probed using an antibody that recognizes the carboxyl terminus of the lactose permease. The amount of lactose permease was then determined by laser densitometry.


RESULTS

Isolation of Site-directed Mutants

As noted above, a conserved motif is found within hydrophilic loop 2/3 and 8/9 among members of the USA superfamily. Fig. 1describes the loop 2/3 motif in 65 members of the USA superfamily. According to an extensive hydropathicity analysis(21) , the first three or four amino acid residues are commonly predicted to lie within transmembrane domain 2. At the first and eighth positions, a glycine residue (small side chain volume, high -turn propensity) is highly conserved although amino acids with a small side chain volume (i.e. alanine or serine) or amino acids with a high or moderate turn propensity (proline or tyrosine) are found in a few cases. Hydrophobic amino acids are found at the second, third, fourth, and seventh positions. An acidic residue is frequently present at the fifth position while basic residues are conserved at the sixth, ninth, and tenth position. It should be noted that an extra amino acid is occasionally ``inserted'' into this decapeptide motif sequence. In the case of the lactose permease, an extra leucine residue is found between the glycine at the eighth position and the arginine at the ninth position.


Figure 1: Alignment of the loop 2/3 domains of the lactose permease and selected members of the USA superfamily. The end of transmembrane segment 2 and the beginning of transmembrane segment 3 are shaded in gray. The members of the superfamily are identical to those described in Ref. 21.



As mentioned above, evidence from the tetracycline antiporter has shown that the conserved glycine residues, acidic residue, and one of the basic residues, are very important for transport activity(26, 27, 28) . To explore whether these conserved residues are also important for the function of the lactose permease, we have made the site-directed mutants described in .

To determine if these mutations affect permease levels of expression, the wild-type and mutant strains were analyzed by Western blotting and densitometry. As shown in , the majority of the mutants showed levels that were similar to the wild-type strain. In most cases, it seems likely the variability among the mutants is due to inaccuracies in this technique rather than actual differences in the amount of permease. However, it was striking that both of the aspartate substitutions at positions 64 and 71 showed very low levels of permease proteins. Therefore, these mutants were not analyzed with regard to transport activity.

Phenotype on MacConkey Plates

The phenotype of bacterial colonies on MacConkey plates can provide qualitative information concerning the functionality of transport. Those cells that are able to appreciably transport and subsequently metabolize the added sugar form red colonies, whereas a defect in this process results in the formation of white colonies. In general, a lactose permease mutant must be greatly defective in transport to prevent a red colony phenotype. When the wild-type and mutant strains were plated on MacConkey plates containing lactose (a -galactoside) or melibiose (an -galactoside), some general trends were observed. As shown in , mutations at the first glycine (i.e. Gly-64 in the lactose permease) or at the fifth acidic residue (Asp-68) were found to be highly defective in sugar transport. A few mutations at the seventh glycine (Gly-71) were slightly defective. Surprisingly, none of the mutants at the basic residues (e.g. Lys-69, Arg-73, or Lys-74) appeared to be defective even though nonbasic substitutions were made at these sites.

In Vitro Galactoside Transport

The experiments of indicate that the first- and fifth-position mutants are severely defective in their ability to transport galactosides while the other mutants are generally less defective or exhibit a wild-type phenotype. In the experiment of I, in vitro transport assays were carried out to provide a more quantitative description of the uptake process. In I, the wild-type and mutant strains were analyzed for their ability to transport [C]lactose. In ``downhill'' transport assays, plasmids containing the wild-type lacY gene or mutant genes were introduced into an E. coli strain that is lacZ (i.e. -galactosidase positive). Upon entry into the cell, lactose is rapidly metabolized so that the extracellular lactose concentration remains higher than the intracellular concentration(39) . Therefore, under these conditions, lactose transport is downhill or with its concentration gradient. In the ``uphill'' transport assays the plasmids were transformed into a bacterial strain that is -galactosidase negative.

As expected from the results of , most of the first position mutants were very defective in both the downhill and uphill transport of [C]lactose (see I). Only the Ala-64 mutant was similar to the wild-type strain in downhill transport and had approximately 50% uphill accumulation. Besides the Ala-64 substitution, the next highest values were observed in the Ser-64 strain which showed transport activities that were less than 10% the wild-type level. These results are consistent with the conservation of residues at this position. Aside from glycine, alanine and serine are the two residues that are most likely to be at the first position in this motif (see Fig. 1).

Very different results were obtained at the eighth-position glycine. With regard to downhill lactose transport, all of the site-directed mutants at this position were observed to have transport levels that are similar to the wild-type strain (see I). These results indicate that, under the conditions of our transport assays (37 °C, pH 7.0), a glycine at the eighth position is not particularly necessary for transport activity per se. Likewise, Gly-71 does not appear essential for the proper insertion and/or stability of the lactose permease since the levels of expression of the position 71 mutants were found to be similar to wild-type (see ). These observations seem particularly surprising considering the extremely high degree of conservation that occurs at the eighth position. However, it remains a possibility that an eighth-position glycine may be important for transport activity and/or expression under conditions that are different from those described in this study. Also, it should be pointed out that some of the mutants, such as Cys-71, Pro-71, and Leu-71, exhibited uphill accumulation levels that were less than 40% that of the wild-type value indicating that substitutions at this position can affect the transport process.

The acidic residue at the fifth position appears to be critical for maximal transport activity. As shown in I, all of the mutants at this position were found to have less than 5% of the downhill rate of lactose transport. The results obtained with the Glu-68 mutant seem particularly striking because it retains the negative carboxyl group and a glutamate is commonly found at the fifth position in the analogous loop 8/9 motif. Nevertheless, the results obtained with the lactose permease indicate a rather strict requirement for an aspartate residue at position 68.

Finally, the role of the basic residues was examined by making single changes at all three basic residues. None of the basic residues were found to be required for normal downhill transport function. These results are quite different from those obtained with the tetracycline antiporter where it was found that the basic residue at the ninth position (analogous to Arg-73 in the lactose permease) is essential for transport activity(27) . Mutations at the basic residues in the lactose permease were shown to have only moderate defects in their uphill accumulation ranging from 50% for the Gly-69 strain to 83% for the Gly-73 mutant (see I).

HTransport

Since the wild-type residue at position 68 is acidic, it was of interest to test whether the position 68 mutants are able to couple H and sugar transport. To accomplish this, a pH electrode was used to measure H transport upon the addition of lactose. These results are shown in Fig. 2. When lactose was added, the wild-type strain exhibited a rapid alkalinization of the medium due to the cotransport of H and lactose into the bacterial cytoplasm. Similarly the Ala-68 and Thr-68 mutant strains also exhibited a significant alkalinization. As expected, the magnitude of the mutant's alkalinization was less than that of the wild-type strain since these mutants have a diminished rate of lactose transport (see I).


DISCUSSION

The conservation of a motif within a large superfamily is indicative of an important functional and/or structural role. With regard to the lactose permease, the experiments described in this study support this notion. The most dramatic results were obtained from mutations at the first-position glycine and the fifth-position aspartate. A variety of single amino acid substitutions were shown to result in substantial or complete loss of transport activity. By comparison, the eighth-position glycine and the three basic residues seem to be less essential although many single mutations at these locations showed significant decreases in the uphill accumulation of lactose. However, the possibility should be considered that these residues may be important for transport activity and/or expression under conditions that are different from the current study.

The role of glycine residues within loop 2/3 may be related to different features of the glycine side chain. In particular, this side chain has the smallest volume and exhibits a high -turn propensity. Either or both of these features may be important with regard to transport function. At the first position, there is evidence to suggest that both features may play a role. In the current and other studies, it appears that only amino acids with a smaller side chain, such as alanine and to a lesser extent serine, are able to substitute for glycine. These results are consistent with the idea that side chain volume plays an important role. This conclusion also correlates with the observation that alanine and serine are the most likely amino acids, other than glycine, to occur at the first position among members of the USA superfamily. However, it is also worthwhile to point out that proline (high -turn propensity) and tyrosine (moderate -turn propensity) are occasionally found at the first position even though these residues have significantly larger side chain volumes. In loop 8/9 of the lactose permease, a proline is found in the first position of this motif. Therefore, it is interesting to speculate that a combination of side chain volume and -turn propensity may contribute to the effects of amino acid substitutions at the first position. In some cases, such as the Pro-64 mutant in the lactose permease, the negative effect of a large side chain volume may override the positive effect of high -turn propensity.

At the eighth position, the role of the glycine side chain remains unclear. Mutations with large side chains and/or low -turn propensity, showed relatively high transport activity. Also, the position 71 mutants with low uphill transport activity (e.g. cysteine, proline, and leucine, see I), do not correlate with high side chain volume or low -turn propensity. Therefore, based solely on these transport experiments, the results do not suggest that side chain volume or -turn propensity are critical at the eighth position. This conclusion seems very unusual considering that the glycine at the eighth position is the most highly conserved amino acid in the loop 2/3 motif and it is 100% conserved in the loop 8/9 motif.

All substitutions of the aspartate at position 68, including the conservative glutamate substitution, produced permeases with substantially lower levels of transport activity. However, it is clear that an acidic residue at position 68 is not essential for sugar transport. The Thr-68, Tyr-68, Ser-68, and Ala-68 mutants were able to catalyze the downhill and uphill accumulation of sugars to a low, but significant level. Likewise, an acidic residue at this position does not appear to be necessary for cation recognition since the nonionizable Ala-68 and Thr-68 mutants were directly shown to transport H upon the addition of lactose. Among the USA superfamily, an aspartate is not always conserved in the loop 2/3 motif. However, it is rather striking that an acidic residue (e.g. aspartate or glutamate) is nearly always present at the fifth position of this motif in, at least, one of the two repeated motifs. The only exception is a gene designated LTP from Leishmania(40) . In any case, it is intriguing that a negative charge in one of the two repeated motifs has been highly conserved throughout evolution. Future work will be necessary to determine whether this negative charge is always an important requirement for transport among this superfamily of proteins.

Since the GXXX(D/E)(R/K)XG(R/K)(R/K) motif is conserved among proteins that transport a variety of different kinds of solutes, it seems unlikely that it is directly involved with solute recognition. Instead of substrate binding, alternative roles for the GXXX(D/E)(R/K)XG(R/K)(R/K) motif can be postulated. For example, since this motif is found along the cytoplasmic domain of transporters, it is interesting to speculate that this region of the protein may be a critical domain that allows access of the solute into the cytoplasm. Along these lines, the conserved motif may be important in facilitating global conformational changes following solute binding. As shown in Fig. 3, the C1 conformation of the permease provides access to solute binding from the outside of the cell while in the C2 conformation the solute has access to the cytoplasm. The loop 2/3 motif may be important in promoting the interconversion between C1 and C2. Another possibility may be that loop 2/3 and/or loop 8/9 may be acting as a gate for channel opening and closing.


Figure 3: Hypothetical role of the loop 2/3 motif in conformational changes of the lactose permease. The three-dimensional arrangement of transmembrane domains has been proposed in Ref. 21. The channel-lining segments are TM-1, TM-2, TM-4, TM-5, TM-7, TM-8, TM-10, TM-11, while the scaffolding segments are TM-3, TM-6, TM-9, and TM-12. The two halves of the protein exhibit rotational symmetry around a central axis. The C1 conformation has its solute binding site accessible from the outside while the same site in the C2 conformation is accessible from the inside.



  
Table: Expression levels in mutant strains


  
Table: Phenotype on MacConkey plates


  
Table: Lactose transport activities of wild-type and mutant strains

[C]Lactose was measured as described under ``Materials and Methods.'' Downhill lactose uptake was carried out in strain HS4006/F`IZY (Ref. 42) containing the lacY plasmids described within the table. Uphill accumulation was carried out in strain, T184 (Ref. 41), that is -galactosidase negative. The values are presented as the % of wild type activity.



FOOTNOTES

*
This work was supported by Grant GM53259 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bioprocess Technical Institute, 240 Gortner Laboratories, 1479 Gortner Ave., St. Paul, MN 55108.

The abbreviation used is: USA, uniport, symport, antiport.


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