Lactose permease H+-lactose symporter: mechanical switch or Brownian ratchet?

Lactose permease structure is deemed consistent with a mechanical switch device for H(+)-coupled symport. Because the crystallography-assigned docking position of thiodigalactoside (TDG) does not make close contact with several amino acids essential for symport; the switch model requires allosteric interactions between the proton and sugar binding sites. The docking program, Autodock 3 reveals other lactose-docking sites. An alternative cotransport mechanism is proposed where His-322 imidazolium, positioned in the central pore equidistant (5-7 A) between six charged amino acids, Arg-302 and Lys-319 opposing Glu-269, Glu-325, Asp-237, and Asp-240, transfers a proton transiently to an H-bonded lactose hydroxyl group. Protonated lactose and its dissociation product H(3)O+ are repelled by reprotonated His-322 and drift in the electrostatic field toward the cytosol. This Brownian ratchet model, unlike the conventional carrier model, accounts for diminished symport by H322N mutant; how H322 mutants become uniporters; why exchanging Lys-319 with Asp-240 paradoxically inactivates symport; how some multiple mutants become revertant transporters; the raised export rate and affinity toward lactose of uncoupled mutants; the altered specificity toward lactose, melibiose, and galactose of some mutants, and the proton dissociation rate of H322 being 100-fold faster than the symport turnover rate.     

A Suppressor Analysis of Residues Involved in Cation Transport in the Lactose Permease: Identification of a Coupling Sensor

Four amino acids critical for lactose permease function were altered using site-directed mutagenesis. The resulting Quad mutant (E269Q/R302L/H322Q/E325Q) was expressed at 60% of wild-type levels but found to have negligible transport activity. The Quad mutant was used as a parental strain to isolate suppressors that regained the ability to ferment the α-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and ΔI298). All of the suppressors transported α-galactosides at substantial rates. In addition, the Q60L, ΔI298 and K319N suppressors regained a small but detectable amount of lactose transport. Assays of sugar-driven cation transport showed that both the Q60L and K319N suppressors couple the influx of melibiose with cations (H⁺ or H₃O⁺). Taken together, the data show that the cation-binding domain in the lactose permease is not a fixed structure as proposed in previous models. Rather, the data are consistent with a model in which several ionizable residues form a dynamic coupling sensor that also may interact directly with the cation and lactose.     

A Triple Mutant, K319N/H322Q/E325Q, of the Lactose Permease Cotransports H+ with Thiodigalactoside

In a previous study, we characterized a lactose permease mutant (K319N/E325Q) that can transport H⁺ ions with sugar. This result was surprising because other studies had suggested that Glu-325 plays an essential role in H⁺ binding. To determine if the lactose permease contains one or more auxiliary H⁺ binding sites, we began with the K319N/E325Q strain, which catalyzes a sugar-dependent H⁺ leak, and isolated third site suppressor mutations that blocked the H⁺ leak. Three types of suppressors were obtained: H322Y, H322R, and M299I. These mutations blocked the H⁺ leak and elevated the apparent K _m value for lactose. The M299I and H322Y suppressors could still transport H⁺ with β-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H⁺/sugar cotransport. Four mutant strains containing a nonionizable substitution at codon 322 (H322Q) were analyzed. None of these were able to catalyze uphill accumulation of lactose, however, all showed some level of substrate-induced proton accumulation. The level seemed to vary based on the substrate being analyzed (lactose or TDG). Most interestingly, a triple mutant, K319N/H322Q/E325Q, catalyzed robust H⁺ transport with TDG. These novel results suggest an alternative mechanism of lactose permease cation binding and transport, possibly involving hydronium ion (H₃O⁺). Received: 6 November 2000/Revised: 23 March 2001     

Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.     

Engineering conformational flexibility in the lactose permease of Escherichia coli: use of glycine-scanning mutagenesis to rescue mutant Glu325-->Asp

Lactose/H(+) symport by lactose permease of Escherichia coli involves interactions between four irreplaceable charged residues in transmembrane helices that play essential roles in H(+) translocation and coupling [Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix IX) with Glu325 (helix X)], as well as Glu126 (helix IV) and Arg144 (helix V) which are obligatory for substrate binding. The conservative mutation Glu325-->Asp causes a 10-fold reduction in the V(max) for active lactose transport and markedly decreased lactose-induced H(+) influx with no effect on exchange or counterflow, neither of which involves H(+) symport. Thus, shortening the side chain may weaken the interaction of the carboxyl group at position 325 with the guanidino group of Arg302. Therefore, Gly-scanning mutagenesis of helices IX and X and the intervening loop was employed systematically with mutant Glu325-->Asp in an effort to rescue function by introducing conformational flexibility between the two helices. Five Gly replacement mutants in the Glu325-->Asp background are identified that exhibit significantly higher transport activity. Furthermore, mutant Val316-->Gly/Glu325-->Asp catalyzes active transport, efflux, and lactose-induced H(+) influx with kinetic properties approaching those of wild-type permease. It is proposed that introduction of conformational flexibility at the interface between helices IX and X improves juxtapositioning between Arg302 and Asp325 during turnover, thereby allowing more effective deprotonation of the permease on the inner surface of the membrane [Sahin-Tóth, M., Karlin, A., and Kaback, H. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10729-10732.     

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.     

Manipulating conformational equilibria in the lactose permease of Escherichia coli.

A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized. Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed. In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored.     

lac permease of Escherichia coli: arginine-302 as a component of the postulated proton relay

The lac permease of Escherichia coli was modified by site-directed mutagenesis such that Arg-302 in putative helix IX was replaced with Leu. In addition, Ser-300 (helix IX) was replaced with Ala, and Lys-319 in putative helix X was replaced with Leu. Permease with Leu at position 302 manifests properties that are similar to those of permease with Arg in place of His-322 [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4483]. Thus, permease with Leu-302 is markedly defective in active lactose transport, efflux, exchange, and counterflow but catalyzes downhill influx of lactose at high substrate concentrations without H+ translocation. In contrast, permease molecules with Ala at position 300 or Leu at position 319 catalyze lactose/H+ symport in a manner indistinguishable from that of wild-type permease. By molecular modeling, Arg-302 may be positioned in helix IX so that it faces the postulated His-322/Glu-325 ion pair in helix X. In this manner, the guanidino group in Arg-302 may interact with the imidazole of His-322 and thereby play a role in the H+ relay suggested to be involved in lactose/H+ symport [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4486].     

Permease on parade: application of site-directed mutagenesis to ion-gradient driven active transport

Oligonucleotide-directed, site-specific mutagenesis is being applied to the problem of ion-gradient driven active transport with the lac permease as a model system. It has been shown that Arg-302, His-322 and Glu-325, neighboring residues in putative transmembrane helices IX and X, play an important role in lactose-coupled H⁺ translocation, possibly as components of a catalytic triad similar to that found in the serine proteases. In addition, Cys residues, long thought to be involved in the mechanism of the permease, are not directly involved in either substrate binding or H⁺ translocation. Finally, a variety of mutations have no effect on permease activity indicating that single amino acid changes do not lead indiscriminately to long-range conformational alterations.     

Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.

Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.     

Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient.

The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322. Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined. Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82). Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold). Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient. The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains. The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant. Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e. melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose). In contrast, the single Val-177 strain exhibited a normal recognition for these sugars.     

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

lac permease with Ala in place of Glu325 was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The reconstituted molecule is completely unable to catalyze lactose/H+ symport but catalyzes exchange and counterflow at least as well as wild-type permease. In addition, Ala325 permease catalyzes downhill lactose influx without concomitant H+ translocation and binds p-nitrophenyl alpha-D-galactopyranoside with a KD only slightly higher than that of wild-type permease. Studies with right-side-out membrane vesicles demonstrate that replacement of Glu325 with Gln, His, Val, Cys, or Trp results in behavior similar to that observed with Ala in place of Glu325. On the other hand, permease with Asp in place of Glu325 catalyzes lactose/H+ symport about 20% as well as wild-type permease. The results indicate that an acidic residue at position 325 is essential for lactose/H+ symport and that hydrogen bonding at this position is insufficient. Taken together with previous results and those presented in the following paper [Lee, J. A., Püttner, I. B., & Kaback, H. R. (1989) Biochemistry (third paper of three in this issue)], the findings are consistent with the idea that Arg302, His322, and Glu325 may be components of a H+ relay system that plays an important role in the coupled translocation of lactose and H+.     

lac permease of Escherichia coli: histidine-322 and glutamic acid-325 may be components of a charge-relay system

When Glu-325 in the lac permease of Escherichia coli is replaced with Ala, lactose/H+ symport is abolished. Thus, the altered permease catalyzes neither uphill lactose accumulation nor efflux. Remarkably, however, permease with Ala-325 catalyzes exchange and counterflow at completely normal rates. Taken together with the results presented in the accompanying paper [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (preceding paper in this issue)], the findings suggest that the His-322 and Glu-325 may be components of a charge-relay system that plays an important role in the coupled translocation of lactose and H+.     

Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues

Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity [Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765], sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional role of arginine 302 within the lactose permease of Escherichia coli.

Within the lactose permease, an arginine residue is found on a transmembrane segment at position 302. Based upon the effects of mutations at or in the vicinity of Arg-302, this residue has been implicated to be involved with H+ and/or sugar recognition. To further elucidate the role of this residue, we have substituted Arg-302 with serine, histidine, and leucine via site-directed mutagenesis. All three of these substitutions result in an impaired ability to transport galactosides as evidenced by their poor growth on minimal plates supplemented with lactose or melibiose. Furthermore, in vitro transport assays revealed substantial alterations in the kinetic constants for downhill lactose transport. The wild-type strain exhibited a Km for lactose transport of 0.30 mM and a Vmax of 267 nmol of lactose/min.mg of protein. The Ser-302, His-302, and Leu-302 were observed to have Km values of 0.18, 2.3, and 2.8 mM, and Vmax values of 11.6, 56.4, and 22.0 nmol of lactose/min.mg of protein, respectively. In uphill transport assays, all three mutants were unable to accumulate beta-methyl-D-thiogalactoside. However, both the Ser-302 and His-302 mutants were able to accumulate lactose against a concentration gradient. During H+ transport assays, all three mutants were shown to transport H+ in conjunction with thiodigalactoside. In addition, the Ser-302 and His-302 strains exhibited small alkalinizations upon the addition of lactose. However, for the Leu-302 mutant, the addition of lactose did not result in a significant level of H+ transport. Finally, experiments were conducted which were aimed at measuring the ability of the mutant permeases to catalyze an H+ leak. In this regard, a comparison was made between the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses. The results of these experiments suggest that mutations at position 302 cause a sugar-dependent H+ leak.     

Bistability of the <Emphasis Type="Italic">lac</Emphasis> Operon During Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> on Lactose and Lactose + Glucose

The lac operon of Escherichia coli can exhibit bistability. Early studies showed that bistability occurs during growth on TMG/succinate and lactose + glucose, but not during growth on lactose. More recently, studies with lacGFP-transfected cells show bistability during growth on TMG/succinate, but not during growth on lactose and lactose + glucose. In the literature, these results are invariably attributed to variations in the destabilizing effect of the positive feedback generated by induction. Specifically, during growth on TMG/succinate, lac induction generates strong positive feedback because the permease stimulates the accumulation of intracellular TMG, which in turn, promotes the synthesis of even more permease. This positive feedback is attenuated during growth on lactose because hydrolysis of intracellular lactose by β-galactosidase suppresses the stimulatory effect of the permease. It is attenuated even more during growth on lactose + glucose because glucose inhibits the uptake of lactose. But it is clear that the stabilizing effect of dilution also changes dramatically as a function of the medium composition. For instance, during growth on TMG/succinate, the dilution rate of lac permease is proportional to its activity, e, because the specific growth rate is independent of e (it is completely determined by the concentration of succinate). However, during growth on lactose, the dilution rate of the permease is proportional to e ² because the specific growth rate is proportional to the specific lactose uptake rate, which in turn, proportional to e. We show that: (a) This dependence on e ² creates such a strong stabilizing effect that bistability is virtually impossible during growth on lactose, even in the face of the intense positive feedback generated by induction. (b) This stabilizing effect is weakened during growth on lactose + glucose because the specific growth rate on glucose is independent of e, so that the dilution rate once again contains a term that is proportional to e. These results imply that the lac operon is much more prone to bistability if the medium contains carbon sources that cannot be metabolized by the lac enzymes, e.g., succinate during growth on TMG/succinate and glucose during growth on lactose + glucose. We discuss the experimental data in the light of these results.     

Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli

Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding. To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made. The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport. The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R. In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max). A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient. Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding. An alternative explanation for their role in maintaining secondary structure is discussed.     

Lactose permease mutants which transport (malto)-oligosaccharides.

Lactose permease mutants, which were previously isolated in sugar specificity studies, were screened for their abilities to transport the trisaccharide maltotriose. Six multiple mutants (e.g., five double mutants and one triple mutant) were identified as forming fermentation-positive colonies on maltotriose MacConkey plates and were also shown to grow on maltotriose minimal plates. All of these multiple mutants contained a combination of two or three amino acid substitutions at position 177, 236, 306, or 322 within the permease. In contrast, none of the corresponding single mutants at these locations were observed to exhibit an enhanced rate of maltotriose transport. In whole-cell assays, the multiple mutants were shown to transport relatively long alpha-nitrophenylglucoside (alpha NPG) molecules. In certain cases, alpha NPG molecules containing up to four glucose residues in addition to the nitrophenyl group were shown to be transported to a significant degree. Overall, the abilities of lactose permease mutants to transport maltotriose and long alpha NPGs are discussed with regard to the dimensions of the sugar and the mechanism of sugar transport.     

Effect of distance and orientation between arginine-302, histidine-322, and glutamate-325 on the activity of lac permease from Escherichia coli

lac permease of Escherichia coli was modified by site-directed mutagenesis in order to investigate the effects of polarity, distance, and orientation between the components of a putative H+ relay system (Arg302/His322/Glu325) postulated to be involved in lactose-coupled H+ translocation. The importance of polarity between His322 and Glu325 was studied by interchanging the residues, and the modified permease--H322E/E325H--is inactive in all modes of translocation. The effect of distance and/or orientation between His322 and Glu325 was investigated by interchanging Glu325 with Val326, thereby moving the carboxylate one residue around putative helix X. The resulting permease molecule--E325V/V326E--is also completely inactive; control mutations, E325V [Carrasco, N., Püttner, I. B., Antes, L. M., Lee, J. A., Larigan, J. D., Lolkema, J. S., Roepe, P. D., & Kaback, H. R. (1989) Biochemistry (second paper of three in this issue)], and E325A/V326E, indicate that a Glu residue at position 326 inactivates the permease. The wild-type orientation between His and Glu was then restored by further mutation of E325V/V326E to introduce a His residue into position 323 or by interchanging Met323 with His322. The resulting permease molecules--M323H/E325V/V326E and H322M/M323H/E325V/V326E--contain the wild-type His/Glu orientation, but the His/Glu ion pair is rotated about the helical axis by 100 degrees relative to Arg302 in putative helix IX. Both mutants are inactive with respect to all modes of translocation. The results provide strong support for the contention that the polarity between His322 and Glu325 and the geometric relationship between Arg302, His322, and Glu325 are critical for permease activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutants of the Lactose Carrier of Escherichia coli Which Show Altered Sugar Recognition Plus a Severe Defect in Sugar Accumulation

Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog β-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K _m for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V _max ) of normal. All of the mutants accumulated methyl-α-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation. Received: 12 October 1999/Revised: 21 December 1999