Purified lac permease and cytochrome o oxidase are functional as monomers

Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination. The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas. In nonenergized proteoliposomes, both proteins appear to reconstitute as monomers based on (i) the variation of intramembrane particle density with protein concentration; (ii) the ratio of particles corresponding to each protein in proteoliposomes reconstituted with a known ratio of permease to oxidase; and (iii) the dimensions of the particles observed in tantalum replicas. The intramembrane particle diameters in tantalum replicas are about 20-25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of lac permease and cytochrome o. The diameters and heights of the permease and cytochrome o in tantalum replicas are 5.1 nm X 2.8 nm and 7.4 nm X 4.2 nm, respectively. Furthermore, a higher percentage of lac permease molecules exhibits a notch or cleft in tantalum replicas relative to platinum/carbon replicas. Importantly, the initial rate of lactose/H+ symport in proteoliposomes varies linearly with the ratio of lac permease to phospholipid, and no change is observed in either the size or distribution of lac permease molecules when the proteoliposomes are energized. The results taken as a whole provide a strong indication that both lac permease and cytochrome o reconstitute into proteoliposomes as monomers, that the permease does not dimerize in the presence of the H+ electrochemical gradient, and that both molecules are completely functional as monomers.     

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+.     

Construction of a functional lactose permease devoid of cysteine residues

By use of oligonucleotide-directed, site-specific mutagenesis, a lactose (lac) permease molecule was constructed in which all eight cysteinyl residues were simultaneously mutagenized (C-less permease). Cys154 was replaced with valine, and Cys117, -148, -176, -234, -333, -353, and -355 were replaced with serine. Remarkably, C-less permease catalyzes lactose accumulation in the presence of a transmembrane proton electrochemical gradient (interior negative and alkaline). Thus, in intact cells and right-side-out membrane vesicles containing comparable amounts of wild-type and Cys-less permease, the mutant protein catalyzes lactose transport at a maximum velocity and to a steady-state level of accumulation of about 35% and 55%, respectively, of wild-type with a similar apparent Km (ca. 0.3 mM). As anticipated, moreover, active lactose transport via C-less permease is completely resistant to inactivation by N-ethylmaleimide. Finally, C-less permease also catalyzes efflux and equilibrium exchange at about 35% of wild-type activity. The results provide definitive evidence that sulfhydryl groups do not play an essential role in the mechanism of lactose/H+ symport. Potential applications of the C-less mutant to studies of static and dynamic aspects of permease structure/function are discussed.     

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].     

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)     

Effect of the lipid phase transition on the lactose permease from Escherichia coli

The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 --> Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H(+) symport, Arrhenius plots with wild-type permease exhibit a discontinuity at 18-19 degrees C with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H(+) symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325 --> Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H(+) translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H(+) translocation rather than the conformational change(s) responsible for translocation across the membrane.     

An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport

The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport. Both mutants were able to transport lactose downhill to a significant degree. The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars. With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree. This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions. Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain. The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant. In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+. The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain. When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane. The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux. The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e. 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth. These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site.     

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.     

Site-specific mutagenesis of cysteine 148 to serine in the lac permease of Escherichia coli

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the lac Y gene of Escherichia coli such that Cys148 in the lac permease is converted to Ser. A mutagenesis protocol is used that significantly improves the efficiency of mutant recovery by in vitro methylation of closed-circular heteroduplex DNA containing the mutation, followed by nicking with HindIII in the presence of ethidium bromide and heat denaturation prior to transfection. In contrast to Gly148 permease (Trumble, W.R., Viitanen, P.V., Sarkar, H.K., Poonian, M.S., and Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860-867), permease containing Ser at position 148 catalyzes active lactose transport at a rate comparable to wild-type permease. Like Gly148 permease, however, transport activity is less sensitive to inactivation by N-ethylmaleimide, and galactosyl-1-thio-beta-D-galactopyranoside affords no protection against inactivation. The observations provide strong support for the contention that Cys148 is obligatory for substrate protection against inactivation by sulfhydryl reagents, but does not play an essential role in lactose:H+ symport.     

Biosensors based on membrane transport proteins.

We propose a novel class of biosensors based on membrane bound receptors or transport proteins as the sensing element. The protein is incorporated in a planar lipid bilayer which covers the transducer. The transducer may detect an electric current, a voltage, or a change in fluorescence. A prototype lactose sensor is presented which consists of a quartz slide covered by a lipid membrane containing the protein lactose permease from Escherichia coli. This protein is a lactose/H+ cotransporter, hence lactose in the external medium initiates lactose/H+ cotransport across the lipid membrane. This leads to a rise in proton concentration in the small volume between the lipid membrane and the quartz surface which can be detected by a pH-sensitive fluorescence dye.     

Role of cysteine residues in the lac permease of Escherichia coli

Oligonucleotide-directed, site-specific mutagenesis has been utilized to replace cysteine residues 117, 333, or 353 and 355 with serine in the lac permease of Escherichia coli. Replacement of Cys-117 or Cys-333 has no significant effect on permease activity, while permease with serine residues in place of Cys-353 and Cys-355 has about 50% of wild-type permease activity. The results provide a clear demonstration that cysteine residues at positions 117, 333, 353, and 355 are not obligatory for lactose/H+ symport. When considered in conjunction with previous findings, the results indicate that, of the eight cysteine residues in the lac permease, only Cys-154 is important for lactose transport. As discussed, the conclusion has important implications for the hypothesis that sulfhydryl-disulfide interconversion plays an important role in the symport mechanism.     

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.     

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.     

What's new with lactose permease

The lactose permease ofEscherichia coli is a paradigm for polytopic membrane transport proteins that transduce free energy stored in an electrochemical ion gradient into work in the form of a concentration gradient. Although the permease consists of 12 hydrophobic transmembrane domains in probable -helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops, little information is available regarding the folded tertiary structure of the molecule. In a recent approach site-directed fluorescence labeling is being used to study proximity relationships in lactose permease. The experiments are based upon site-directed pyrene labeling of combinations of paired Cys replacements in a mutant devoid of Cys residues. Since pyrene exhibits excimer fluorescence if two molecules are within about 3.5Å, the proximity between paired labeled residues can be determined. The results demonstrate that putative helices VIII and IX are close to helix X. Taken together with other findings indicating that helix VII is close to helices X and XI, the data lead to a model that describes the packing of helices VII to XI.K. Jung, H. Jung and G. G. Privé are Postdoctoral Fellows of the Deutscher Akademischer Austauschdienst, the European Molecular Biology Organization, and the American Cancer Society (California Division), respectively.     

Secondary active transport

Secondary active transport is defined as the transport of a solute in the direction of its increasing electrochemical potential coupled to the facilitated diffusion of a second solute (usually an ion) in the direction of its decreasing electrochemical potential. The coupling agents are membrane proteins (carriers), each of which catalyzes simultaneously the facilitated diffusion of the driving ion and the active transport of a given solute. The review starts with some considerations on the energetics followed by a presentation of the kinetics of secondary active transport. Examples of information which may be gained by such studies are discussed. In the second part, some examples of secondary transport are given; we also describe the characteristics of the corresponding carriers. The various transport systems presented are: the D-glucose/Na+ symport in brush-border membranes, the lactose/H+ symport in E. coli, the Na+/H+ antiport, the different transport systems in the inner mitochondrial membrane.     

Substitution of glutamine-60 with glutamic acid causes the lac permease of Escherichia coli to become temperature sensitive

The lac Y gene of Escherichia coli was modified by oligonucleotide-directed, site-specific mutagenesis so that Gln-60 is replaced with Glu. Although the replacement introduces a negative charge into a putative hydrophobic, transmembrane alpha-helical segment of the lac permease, lactose/H+ symport is unimpaired. However, the modified permease is more susceptible to heat inactivation. That is, upon incubation at 45 degrees C, Glu-60 permease loses activity with a t1/2 of 20 min relative to a t1/2 of 50 min with wild-type permease.     

Site-directed mutagenesis of Pro327 in the lac permease of Escherichia coli

By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease. Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease. Finally, permease with Leu at position 327 is completely inactive. The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327. It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important.     

Facilitated diffusion properties of melibiose permease in Escherichia coli membrane vesicles. Release of co-substrates is rate limiting for permease cycling

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+). Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+. The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes. This suggests that the permease functions asymmetrically. Cross-comparison between the rates of net [3H]melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease. The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling. This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation. Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.     

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.