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+.     

Conservation of residues involved in sugar/H(+) symport by the sucrose permease of Escherichia coli relative to lactose permease

Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB. Moreover, despite only 28% sequence identity and a marked difference in substrate specificity, the structural organization of the residues involved in sugar-binding and H(+) translocation is conserved in CscB. Functional analyses of mutants in the homologous key residues provide strong evidence that they play a similar critical role in the mechanisms of CscB and LacY.     

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)     

Topography of lactose permease from Escherichia coli

The topography of lactose permease, in native membrane vesicles and after reconstitution of the purified protein into proteoliposomes, has been investigated by labeling the membrane-embedded portions of the protein using photoactivatable, hydrophobic reagents and by labeling the exposed portions of the protein with water-soluble, electrophilic reagents. Some sites of modification have been localized in fragments of the protein produced by chemical and enzymatic cleavage. These define a number of hydrophilic loops and membrane-spanning regions and give some substance to topographic models of the permease. The N-terminal third of the molecule was labeled by three photoactivatable reagents (3-(trifluoromethyl)-3-m-iodophenyldiazirine and the phospholipid analogues 2-(aceto-(4-benzoylphenylether]-1-palmitoylphosphatidylcholine) and 2-(4-azido-2-nitrophenylaminoacetyl)-1-palmitoylphosphatidylcholin e) as well as the water soluble, electrophilic reagents. The C-terminal part of the molecule is labeled by the diazirine and, to a lesser extent, by the phospholipid analogues. It apparently has more nucleophilic groups accessible to water-soluble reagents than the N-terminal domain, in which the density of apparently unreactive ionizable residues proved to be unexpectedly high. The apparent lack of reactivity of some of these residues may be explained either by their being buried in the protein moiety within the membrane domain, or by their close association with other ionizable residues on the surface of the protein.     

Two-dimensional crystallization of Escherichia coli lactose permease

A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed. The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers.     

The effects of pH on proton sugar symport activity of the lactose permease purified from Escherichia coli

The lactose permease, which catalyzes galactoside-proton symport into Escherichia coli, has been purified and reconstituted in active form into artificial lipid vesicles. The roles of many detergents and phospholipids in solubilization and stabilization of the activity of the permease have been examined with a view to its eventual crystallization. Initial rates of uptake into reconstituted proteoliposomes determined by rapid mixing techniques proved that the activity of the permease can be comparable to that observed in the intact cell, while the best values for uptake rates obtained with conventional techniques were comparable to those reported for vesicles. The activity of the purified protein has been monitored over time periods of hours to weeks. It is shown that, under the best current conditions, the permease retains full activity for 1 to 2 weeks. Although this is still marginal for its crystallization, future improvements can now be assayed by rather stringent criteria. The mechanism of galactoside transport into reconstituted proteoliposome has been investigated by examining the effects of pH on influx into the vesicles. It is shown that the observed effects are entirely consistent with the predictions of a simple model of proton symport. The apparent increase in rate of uptake that is observed in the presence of a pH gradient is not so much due to an acceleration by a component of the protonmotive force as to the relaxation of inhibition by a product (internal protons) of the symport reaction.     

Limited proteolysis of lactose permease from Escherichia coli

Escherichia coli lactose permease (also referred to as lactose carrier) is an integral protein of the cytoplasmic membrane. Using lactose permease either radiolabeled biosynthetically in plasmid-bearing E. coli minicells or radioalkylated post-synthetically by chemical modification, we have determined sites on the membrane-bound protein accessible to proteolytic attack and we have characterized several high-molecular-mass products. The most prominent polypeptide obtained from lactose permease radiolabeled biosynthetically is observed after digestion with different proteases. The fragment produced by thermolysin was shown to contain the intact N-terminus and to extend into the region around amino acid residue 140 which, according to secondary structure models, is presumed to be less tightly folded than the rest of the molecule. Evidence is presented that the corresponding fragments obtained after digestion with several other proteases also originate from the N-terminal part of the protein. This N-terminal segment of the lactose carrier is resistant to proteolytic digestion even in the presence of non-ionic detergents and it may represent a tightly folded domain. Additional proteolytic cleavage sites located C-terminal of the Cys148 residue can be inferred.     

Membrane assembly of lactose permease of Escherichia coli.

Lactose permease, the lacY gene product in Escherichia coli, is an integral membrane protein. Its induction was examined in secAts and secYts mutants by measuring o-nitrophenyl-beta-galactoside uptake activity. In contrast to the synthesis of the maltose binding protein, the malE gene product, which is dependent on the secA and secY gene products, lactose permease seemed to be produced and integrated functionally into membrane independently of SecA or SecY. Gene fusion of the lamB signal sequence to the N-terminal part of the lactose permease gene resulted in production of active fused permease in the E. coli membrane. The signal sequence did not seem to be processed, judging from its mobility on SDS polyacrylamide gel electrophoresis. E. coli cell growth was super-sensitive to induction of production of the fused permease with the signal sequence in contrast to induction of the normal lactose permease. These results are consistent with the above observation that production and integration of LacY protein into membrane is relatively independent of the SecY protein that may have a certain specificity for the signal sequence or, more generally, membrane translocation intermediates.     

Binding of p-nitrophenyl alpha-D-galactopyranoside to lac permease of Escherichia coli

Binding of the substrate analogue p-nitrophenyl alpha-D-galactopyranoside (NPG) to lac permease of Escherichia coli in different membrane preparations was investigated. Binding was assayed with an improved version of the centrifugation technique introduced by Kennedy et al. [Kennedy, E.P., Rumley, M.V., Armstrong, J.B. (1974) J. Biol. Chem. 249, 33-37]. Two binding sites for NPG were found with dissociation constants of about 16 microM and 1.6 mM at pH 7.5 and room temperature. With purified lac permease reconstituted into proteoliposomes, it could be shown that one permease molecule binds two substrate molecules. Oxidation of lac permease with the lipophilic quinone plumbagin or alkylation with the sulfhydryl reagent N-ethylmaleimide caused a 12-fold increase in the first dissociation constant. The second dissociation constant seemed to be increased as well, but its value could not reliably be estimated. Ethoxyformylation of lac permease with diethyl pyrocarbonate totally abolished NPG binding. The implications of these results for the catalytic performance of the enzyme are discussed.     

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.     

Sugar recognition by the lactose permease of Escherichia coli

Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.     

cys154 Is important for lac permease activity in Escherichia coli

The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport.     

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.     

Counter-transport mediated by the lactose permease of Escherichia coli.

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of preloaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H+ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide m chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP+). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP+ had no inhibitory effect on the overshoot at pH 6 and below.     

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.     

The lactose permease of Escherichia coli: evidence in favor of a dimer

Lactose permease from Escherichia coli T 206 was purified in octyl-beta-D-glucopyranoside (octyl-glucoside) according to Newman et al. [J. Biol. Chem. (1981) 256, 11804-11808]. In this detergent the protein has a very high tendency to aggregate nonspecifically. Therefore, exchange of octyl-glucoside was performed for another nonionic detergent, dodecyl octaethylene glycol monoether (C12E8), in which the protein is more stable. The amounts of bound C12E8 and phospholipids were measured using radioactive detergent and gas chromatography, respectively, and were found to be respectively 0.2 and 0.15 g/g protein. Analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium) and gel filtration (conventional and high performance liquid chromatography) experiments indicated that in this detergent the lactose permease existed mainly as a dimer. This result is at variance with the monomeric state of the protein reported by Wright et al. [FEBS Lett. (1983) 162, 11-15] in another nonionic detergent (dodecyl-o-beta-maltoside). We discuss the possible reason for this discrepancy and suggest that the dimeric state of association may well reflect the situation that prevails in the membrane.