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.     

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.     

Site-directed mutagenesis of cysteine-148 in the lac permease of Escherichia coli: effect on transport, binding, and sulfhydryl inactivation

By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue [Trumble, W. R., Viitanen, P. V., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860]. Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E. coli T184. Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl alpha-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease. In contrast, the Vmax for lactose transport in vesicles containing mutant permease is significantly decreased. Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl alpha-D-galactopyranoside with high affinity. Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity. Importantly, beta-galactosyl 1-thio-beta-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant. The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease. Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl alpha-D-galactopyranoside from wild-type permease, does not affect binding in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of tyrosine residues in the lac permease of Escherichia coli

By using oligonucleotide-directed, site-specific mutagenesis, each of the 14 Tyr residues in the lac permease of Escherichia coli was replaced with Phe, and the activity of each mutant was studied with respect to active transport, equilibrium exchange, and efflux. Ten of the mutations have no significant effect on permease activity. Of the four mutations that alter activity, replacement of Tyr26 or Tyr336 with Phe severely decreases all modes of translocation, and the binding affinity of the mutant permease for p-nitrophenyl alpha-D-galactopyranoside is markedly decreased (i.e., KD is increased). In addition, the Phe336 mutant permease is inserted into the membrane to a lesser extent than wild-type permease, as judged by immunoblot experiments. Permease containing Phe in place of Tyr236 catalyzes lactose exchange approximately 40% as well as wild-type permease but does not catalyze active transport or efflux. Finally, permease with Phe in place of Tyr382 catalyzes equilibrium exchange normally, but exhibits low rates of active transport and efflux without being uncoupled, thereby suggesting that replacement of Tyr382 with Phe alters a kinetic step involving translocation of the unloaded permease across the membrane.     

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.     

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.     

Functional conservation in the putative substrate binding site of the sucrose permease from Escherichia coli

The sucrose (CscB) permease is the only member of the oligosaccharide:H(+) symporter family in the Major Facilitator Superfamily that transports sucrose but not lactose or other galactosides. In lactose permease (lac permease), the most studied member of the family, three residues have been shown to participate in galactoside binding: Cys148 hydrophobically interacts with the galactosyl ring, while Glu126 and Arg144 are charge paired and form H-bonds with specific galactosyl OH groups. In the present study, the role of the corresponding residues in sucrose permease, Asp126, Arg144, and Ser148, is investigated using a functional Cys-less mutant (see preceding paper). Replacement of Ser148 with Cys has no significant effect on transport activity or expression, but transport becomes highly sensitive to the sulfhydryl reagent N-ethylmaleimide (NEM) in a manner similar to that of lac permease. However, in contrast to lac permease, substrate affords no protection whatsoever against NEM inactivation of transport or alkylation with [(14)C]NEM. Neutral (Ala, Cys) mutations of Asp126 and Arg144 abolish sucrose transport, while membrane expression is not affected. Similarly, combination of two Ala mutations within the same molecule (Asp126-->Ala/Arg144-->Ala) yields normally expressed, but completely inactive permease. Conservative replacements result in highly active molecules: Asp126-->Glu permease catalyzes sucrose transport comparable to Cys-less permease, while mutant Arg144-->Lys exhibits decreased but significant activity. The observations demonstrate that charge pair Asp126-Arg144 plays an essential role in sucrose transport and suggest that the overall architecture of the substrate binding sites is conserved between sucrose and lac permeases.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

The role of protons in the mechanism of galactoside transport via the lactose permease of Escherichia coli

The kinetic mechanism of lactose transport across the cytoplasmic membrane has been investigated and the results related to standard models for the lactose-H+ symport reaction using computer simulation. It is shown that the biphasic kinetics reported for lactose uptake (Kaczorowski, G.J. and Kaback, H.R. (1979) Biochemistry 18, 3691-3697) are consistent with random binding of lactose and protons and rapid subsequent translocation of the ternary lactose-H+-permease complex. Such a model is also shown to explain the observed dependence of the kinetic parameters on the magnitude of the protonmotive force. Both sugar and protons are shown to cause product inhibition of lactose flux and the ability of standard models to account for the pattern of inhibition is discussed. Three apparent dissociation constants have been determined for the protonation reactions in the external medium: two (pKa 6.3 and 9.6) control the activity of the permease, whilst the third (pKa 8.3) controls the affinity of the permease for galactosides. A similar set of dissociation constants has been determined for the internal reactions. Again two (pKa 6 and 9.8) control activity and a third (pKa 8.8) controls the affinity for galactosides. The dissociation reactions characterised by pKa 8.3, 8.8, 9.6 and 9.8 are attributed to the dissociation of the substrate (symported) proton from the binary proton-permease complexes (pKa 8.3 and 8.8) and the ternary proton-galactoside-permease complexes (pKa 9.6 and 9.8). The third pair (pKa 6.3 and 6.0) must be interpreted as describing a separate protonation reaction which may have a regulatory or auxiliary role in transport.     

Characterization and sequencing of the lac Y54-41 "uncoupled" mutant of the lactose permease

The Escherichia coli strain carrying the lac Y54-41 gene encodes a mutant lactose permease which carries out normal downhill transport of galactosides but is defective in uphill accumulation. In this study, the mutant lac Y54-41 gene was cloned onto the multicopy vector pUR270. As expected, the cloned gene was shown to express normal downhill transport activity but was markedly defective in the uphill transport of methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-beta-D-thiogalactopyranoside but at a significantly reduced capacity compared to the wild-type strain. However, under conditions where the mutant and wild-type strains both transport lactose at similar rates, no detectable H+ transport was observed in the mutant strain. The entire cloned lac Y54-41 gene was subjected to DNA sequencing, and a single base substitution was found which replaces glycine 262 in the protein with a cysteine residue. Inhibition experiments showed that the mutant permease is dramatically more sensitive to three different sulfhydryl reagents: N-ethylmaleimide, p-hydroxymericuribenzoate, and p-hydroxymercuriphenylsulfonic acid. However, the lactose analogue, thiodigalactoside, was only marginally effective at protecting against inhibition in the mutant strain. The results are consistent with the idea that the sulfhydryl reagents are inhibiting the mutant permease activity by reacting with cysteine 262.     

Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ～1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ～1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ～500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.     

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies

The effects of various monoclonal antibodies against purified lac carrier protein on carrier-mediated lactose transport were studied in right-side-out membrane vesicles and in proteoliposomes reconstituted with purified lac carrier protein. Out of more than 60 monoclonal antibodies tested, only one antibody, designated 4B1, inhibits transport. Furthermore, the nature of the inhibition is highly specific in that the antibody inhibits only those transport reactions that involve net proton translocation (i.e., active transport, carrier-mediated influx and efflux under nonenergized conditions, and lactone-induced proton influx). In contrast, the antibody has little effect on equilibrium exchange and no effect on generation of the proton electrochemical gradient or on the ability of the carrier to bind a high-affinity ligand. Clearly, therefore, the antibody alters the relationship between lactose and proton translocation at the level of the lac carrier protein. When entrance counterflow is studied with external [1-14C]lactose at saturating and subsaturating concentrations, it is apparent that antibody 4B1 mimics the effects of deuterium oxide [Viitanen, P., Garcia, M.L., Foster, D.L., Kaczorowski, G. J., & Kaback, H.R. (1983) Biochemistry 22, 2531]. That is, the antibody has no effect on the rate or extent of counterflow when external lactose is saturating but stimulates the efficiency of counterflow when external lactose is below the apparent Km. It seems likely, therefore, that the antibody either inhibits the rate of deprotonation or alters the equilibrium between protonated and deprotonated forms of the carrier. Monovalent Fab fragments prepared from antibody 4B1 inhibit transport in a manner that is similar qualitatively to that of the intact antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

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].     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

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.     

Proposed partial beta-structures for lac permease and the Na+/H+ antiporter which use similar transport and H+ coupling mechanisms.

Most antiporters, symporters, and transporters have been represented as containing ten to 14 transmembrane helices, primarily on the basis of hydropathy plots. However, multihelix systems provide no obvious mechanism of transport and no simple way of distinguishing substrates. The models of lac permease and the Na+/H+ antiporter presented here postulate that beta-structures are involved in the transport of substrate, and in following this postulate arrive at readily understandable mechanisms for transport and for substrate specificity. The percentage of beta-structures necessary for these models is low enough that it is not in conflict with prior physical evidence for secondary structures. Immunological data also cannot rule these beta-structure mechanisms invalid. In lac permease the new model is obtained by formal representation of the C-terminal amino acids 243-405 as beta-strands. This formal representation nets two interchangeable beta-barrels which provide a simple mechanism for sugar transport. The alternating barrel system may comprise as little as 1/5 the entire permease. In one configuration the barrel forms a pocket with hydrogen bonding residues oriented to the outside of the cell. In the other configuration the barrel forms an analogous pocket oriented towards the inside. Six particular amino acids participate in the substrate hydrogen bonding schemes of both forms, providing a mechanism to shuttle lactose from the outside to the inside or vice versa. A trigger for change of forms which could couple the beta-barrel to H(+)-transport is easily devised, and it involves the apparently critical His322-Glu325 charge relay system. The Na+/H+ antiporter can be organized similarly with an interchanging beta-barrel-beta-clamshell structure attached to 7-transmembrane helices. Charged amino acid sidechains form the basis of an ionic shuttle which is analogous to the lactose shuttle. In this case, too, coupling of Na+ transport to H+ transport may be accomplished by a histidine-glutamate charge relay system.     

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.     

A high affinity fungal nitrate carrier with two transport mechanisms

We have expressed the CRNA high affinity nitrate transporter from Emericella (Aspergillus) nidulans in Xenopus oocytes and used electrophysiology to study its properties. This method was used because there are no convenient radiolabeled substrates for the transporter. Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the gene was originally identified by chlorate selection there was no evidence for transport of this anion. The gene selection is explained by the high affinity of the transporter for chlorite, and the fact that this ion contaminates solutions of chlorate. The pH-dependence of the anion-elicited currents was consistent with H(+)-coupled mechanism of transport. At any given voltage, currents showed hyperbolic kinetics with respect to extracellular H(+), and these data could be fitted with a Michaelis-Menten relationship. But this equation did not adequately describe transport of the anion substrates. At higher concentrations of the anion substrates and more negative membrane voltages, the currents were decreased, but this effect was independent of changes in external pH. These more complicated kinetics could be fit by an equation containing two Michaelis-Menten terms. The substrate inhibition of the currents could be explained by a transport reaction cycle that included two routes for the transfer of nitrate across the membrane, one on the empty carrier and the other proton coupled. The model predicts that the substrate inhibition of transporter current depends on the cytosolic nitrate concentration. This is the first time a high affinity nitrate transport activity has been characterized in a heterologous system and the measurements show how the properties of the CRNA transporter are modified by changes in the membrane potential, external pH, and nitrate concentration. The physiological significance of these observations is discussed.     

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+.