Reconstitution of the melibiose carrier of Escherichia coli

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.     

An investigation of cysteine mutants on the cytoplasmic loop X/XI in the melibiose transporter of Escherichia coli by using thiol reagents: implication of structural conservation of charged residues

The melibiose transporter (Mel B) of Escherichia coli is a cation-coupled (H(+), Li(+), and Na(+)) membrane protein (MW 50 kDa) consisting of 12 transmembrane helices that are connected by periplasmic and cytoplasmic loops, with both the C- and N-ends located on the cytoplasmic side of the membrane. Previous investigations on the largest cytoplasmic loop X/XI indicated that it is a functional re-entrant loop. In this communication, the cysteine mutants on loop X/XI were studied with charged thiol reagents MTSES, MTSET, and IAA for both the inhibition patterns and charge replacement/function rescue of inactive mutants in which the original charged residues were replaced by neutral cysteines. Strong inhibitions were observed in T373C and V376C by both MTSES and MTSET, consistent with previous results of PCMBS inhibition. The thiol reagents failed to recover the activities of inactive mutants D351C, D354C, and R363C and to inhibit active mutants E357C, K359C, and E365C to any significant extent, suggesting a structural conservation at D351, D354, and R363 and tolerance of structural variations at E357, K359, and E365. The results are consistent with previous observation of structural conservation of functionally charged residues in the transmembrane domains and extend to a loop the contention that in the melibiose transporter functionally important charged residues are structurally conserved.     

Carboxyl-terminal truncations of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is predicted to possess a short NH2 terminus, 11 transmembrane segments joined by short hydrophilic regions, and a 40-residue hydrophilic carboxyl terminus of unknown function. This paper describes truncations of the carboxyl terminus at eight locations using site-specific mutagenesis to introduce stop codons. Measurement of sugar transport and cation-coupling characteristics indicate that the carboxyl tail plays no direct role in substrate recognition or energy transduction. Thirty-six amino acids could be removed from the hydrophilic carboxyl domain without the loss of sugar specificity, facilitated diffusion, uphill transport, H+-coupling or Na+-coupling characteristics. These results are consistent with the hypothesis that the sugar/cation binding site is formed by the interaction of the transmembrane helices 3, 4, 6, 9, and 10 and does not involve the carboxyl-terminal portion of the protein. When truncations were made within the hydrophobic domain of transmembrane helix 11 (truncations of 41 or more residues), the carrier was no longer found in the membrane. This suggests that the carboxyl terminus may be involved in the membrane insertion process, stabilization of the carrier within the membrane following insertion, or protection of the inserted carrier from proteolytic scavenging. A new plasmid that expresses the temperature-resistant isoform of the melibiose carrier under inducible control of a tac promoter, designated pKKMB, is also described.     

Physiological evidence for an interaction between helices II and XI in the melibiose carrier of Escherichia coli

The melibiose carrier from Escherichia coli is a cation-substrate cotransporter that catalyzes the accumulation of galactosides at the expense of H(+), Na(+), or Li(+) electrochemical gradients. Charged residues on transmembrane domains in the amino-terminal portion of this carrier play an important role in the recognition of cations, while the carboxyl portion of the protein seems to be important for sugar recognition. In the present study, we substituted Lys-377 on helix XI with Val. This mutant carrier, K377V, had reduced melibiose transport activity. We subsequently used this mutant for the isolation of functional second-site revertants. Revertant strains showed the additional substitutions of Val or Asn for Asp-59 (helix II), or Leu for Phe-20 (helix I). Isolation of revertant strains where both Lys-377 and Asp-59 are substituted with neutral residues suggested the possibility that a salt bridge exists between helix II and helix XI. To further test this idea, we constructed three additional site-directed mutants: Asp-59-->Lys (D59K), Lys-377-->Asp (K377D), and a double mutant, Asp-59-->Lys/Lys-377-->Asp (D59K/K377D), in which the position of these charges was exchanged. K377D accumulated melibiose only marginally while D59K could not accumulate. However, the D59K/K377D double mutant accumulated melibiose to a modest level although this activity was no longer stimulated by Na(+). We suggest that Asp-59 and Lys-377 interact via a salt bridge that brings helix II and helix XI close to one another in the three-dimensional structure of the carrier.     

Membrane topology of the melibiose carrier of Escherichia coli.

The minimum structural information necessary to formulate and assess mechanistic models of integral membrane protein function is that of membrane topology. This paper characterizes the topological structure of the melibiose carrier of Escherichia coli based on constraints provided by genetic fusions to the compartment-specific reporter protein alkaline phosphatase. Twenty-eight unique chimeras exhibiting either low alkaline phosphatase activity (cytoplasmic location of the fusion joint) or high alkaline phosphatase activity (periplasmic location of the fusion joint) were characterized and used in conjunction with Goldman-Engelman-Steitz hydropathy analysis to model topological structure. The melibiose carrier is predicted to have a cytoplasmic amino terminus, two sets of six transmembrane domains separated by an unusually large cytoplasmic loop ("six-loop-six" arrangement), and a 45-residue cytoplasmic carboxyl tail. Remarkably, the identical six-loop-six arrangement is predicted from the hydrophobicity plots of the H(+)-coupled lactose, arabinose, xylose, and citrate cotransporters of E. coli, the glucose transporter from rat brain, the family of glucose transporters isolated from various human tissues and cell lines, and the human, mouse, and hamster multidrug resistance transporters (Henderson, P.J.F. (1990) Res. Microbiol. 141, 316-328; Maloney, P.C. (1990) Res. Microbiol. 141, 374-383). Such a broad degree of conservation (or convergence) suggests a distinct structural and/or mechanistic advantage associated with the six-loop-six motif. The nature of this advantage is as yet unknown.     

The proximity between helix I and helix XI in the melibiose carrier of Escherichia coli as determined by cross-linking

The melibiose carrier of Escherichia coli is a transmembrane protein that comprises 12 transmembrane helices connected by periplasmic and cytoplasmic loops, with both the N- and C-termini located on the cytoplasmic side. Our previous studies of second-site revertants suggested proximity between several helices, including helices XI and I. In this study, we constructed six double cysteine mutants, each having one cysteine in helix I and the other in helix XI: three mutants, K18C/S380C, D19C/S380C, and F20C/S380C, have their cysteine pairs near the cytoplasmic side of the carrier, and the other three, T34C/G395C, D35C/G395C, and V36C/G395C, have their cysteine pairs near the periplasmic side. In the absence of substrate, disulfide formations catalyzed by iodine and copper-(1,10-phenanthroline)(3) indicate that helix I and helix XI are in immediate proximity to each other on the periplasmic side but not on the cytoplasmic side, as shown by protease cleavage analyses. We infer that the two helices are tilted with respect to each other, with the periplasmic sides in close proximity.     

Cation specificity for sugar substrates of the melibiose carrier in Escherichia coli

A study has been made of the sugar substrate specificities and the cation specificities of the melibiose transport system of Escherichia coli. The following beta-galactosides were found to be transported: lactose, L-arabinose-beta-D-galactoside, D-fructose-beta-D-galactoside, o- and p-nitrophenyl-beta-D-galactosides. These beta-galactosides were cotransported with Na+ but not with H+. The alpha-galactosides raffinose, melibiose and p-nitrophenyl-alpha-galactoside were transported with either H+ or Na+. Of the monosaccharides tested D-galactose could use either Na+ or H+ for cotransport whereas D-fucose, L-arabinose and D-galactosamine could use only Na+. The sugar specificity requirements for H+ cotransport are therefore more exacting than those for Na+ cotransport.     

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.     

H + -substrate cotransport by the melibiose membrane carrier in Escherichia coli

Proton entry into anaerobic Escherichia coli in response to the addition of HCl was measured by monitoring pH changes in the external solution. Preincubation of cells in a Na+ -free medium containing melibiose or methyl-alpha-galactoside (alpha MG) stimulated the rate of H+ entry in response to the acid pulse. This melibiose- or alpha MG-dependent proton pathway appeared to be identical to the melibiose carrier, since the channel was only observed in melibiose-induced cells. Furthermore, this membrane pathway for protons showed the same temperature sensitivity as the melibiose carrier (active at 30 degrees but inactive at 37 degrees). These observations are consistent with the idea that the melibiose transport system provides a pathway for protons in the presence of appropriate substrates, but that the pathway is closed to protons in the absence of the sugar. Such observations indicate that there is an obligatory coupling between H+ flux and melibiose or alpha MG flux through the carrier when Na+ is omitted from the incubation medium.     

Peptide-specific antibody for the melibiose carrier of Escherichia coli localizes the carboxyl terminus to the cytoplasmic face of the membrane

The synthetic decapeptide NH2-Cys-Val-Gly-Ala-Val-Ser-Asp-Val-Lys-Ala-COOH (designated MBct10), which corresponds to the carboxyl terminus of the melibiose carrier of Escherichia coli, was synthesized and used to raise antibodies in a rabbit. Anti-MBct10 antibodies recognizes the normal melibiose carrier but not a truncated carrier lacking 14 carboxyl-terminal amino acids. Thus the antibodies are specific for the carboxyl terminus of the carrier and not for other domains of the protein. When right-side-out and inside-out membrane vesicles were probed with anti-MBct10 serum, only the inside-out vesicles bound antibody. The carboxyl terminus of the melibiose carrier protein is therefore exposed on the cytoplasmic surface of the membrane. The co-localization of both NH2- and carboxyl termini to the cytoplasmic surface dictates that the protein cross the membrane an even number of times. These data together with hydrophobicity analysis support a topological model for the melibiose carrier with 10 or 12 transmembrane domains.     

Sugar recognition mutants of the melibiose carrier of Escherichia coli: possible structural information concerning the arrangement of membrane-bound helices and sugar/cation recognition site

Melibiose carrier mutants, isolated by growing cells on melibiose plus the non-metabolizable competitive inhibitor thiomethyl-beta-galactoside (TMG), were studied to determine sugar and cation recognition abnormalities. Most of the mutants show good transport of melibiose but have lost the recognition of TMG. In addition, most mutants show little or no transport of lactose. Cation recognition is also affected as all of these mutants have lost the ability to transport protons with melibiose. The amino acids causing these mutations were determined by sequencing the melB gene on the plasmid. The mutations were located on helices I, IV, VII, X and XI. We propose that these five helices are in proximity with each other and that they line the sugar/cation transport channel.     

Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli

We isolated mutants of Escherichia coli which showed Li+-resistant growth on melibiose. The melibiose carrier of the mutants lost the ability to couple to H+, whereas it retained the ability to couple to Na+. The mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined. The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier: Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val. These amino acid residues are located in slightly hydrophobic regions of the melibiose carrier. The results provide strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H+ (or Li+) recognition or H+ (or Li+) transport by the melibiose carrier.     

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H⁺, Na⁺, or Li⁺. In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22–100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes. Received: 6 March 2001/Revised: 14 May 2001     

Mutations that simultaneously alter both sugar and cation specificity in the melibiose carrier of Escherichia coli

The isolation and deduced amino acid sequence of 70 melibiose carrier mutants with impaired methyl-beta-D-galactopyranoside (TMG) and cation recognition properties is described. The Km for TMG transport ranged from 1 to greater than 100 mM. Amino acid substitutions occurred at 23 unique sites within the protein. These sites were clustered into four distinct regions: Asp-15 through Ile-18 (cluster I), Tyr-116 through Pro-122 (cluster II), Val-342 through Ile-348 (cluster III), and Ala-364 through Gly-374. Only two sites fell outside of these clusters: Ile-61 and Ala-236. In the native conformation, some or all of these clusters may interact to form the substrate recognition site. Impairment of TMG recognition was accompanied by decreased Li+ inhibition of melibiose transport in all but one mutant. That changes in sugar recognition properties should so frequently accompany changes in cation recognition properties suggests an interaction between the two substrates. A model for such interaction is proposed.     

Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+). This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS). Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site revertants were isolated from K18C and Y31C. The revertants were found to have mutations in helices I, IV, and VII.     

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.     

Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains

Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asp-51 and Asp-120 are important for the transport function of the Escherichia coli melibiose carrier.

Asp-51 and Asp-120 of the Escherichia coli melibiose carrier on plasmid pKKMB were separately replaced by amber codons and transformed into eight amber suppressor strains, producing eight amino acid substitutions for each site. Glu-51 and Glu-120 were the only replacements in the carrier that allowed the cells to ferment melibiose and that showed transport of melibiose against a concentration gradient. Revertants to Glu-51 and Glu-120 show less activity than the wild type. The Asp-51 position is more crucial for Na(+)-stimulated melibiose accumulation than is the Asp-120 site.     

Mechanism of the melibiose porter in membrane vesicles of Escherichia coli

The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux. As opposed to the proton--beta-galactoside symport system [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry 18, 3691], efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane. Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge. Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process. It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.