Carboxyl-terminal Truncations of the Melibiose Carrier of Escherichia coZi*

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 car- boxyl 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 sugarlcation binding site is formed by the interaction of the transmem- brane 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, desig- nated pKKMB, is is also described.

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein which mediates the cotransport of a-and @-galactosides with monovalent cations (see Ref. 1 for a review). Although most cotransport systems of bacteria utilize H' gradients (2), the melibiose carrier is unusual in its ability to use H+, Na+, or even Li' as the coupled cations (3)(4)(5)(6)(7). From an evolutionary perspective, this carrier may thus represent a descendant of the class of transport proteins that bridged the gap between the "H+ economy" of primitive cells and the "Na' economy" of animal cells (8,9). Insights into the structure/function relationships of the melibiose carrier may help to elucidate motifs that are essential to the molecular mechanisms of both prokaryotic and eukaryotic transport proteins.
The gene encoding the melibiose carrier (rnelB) has been cloned and sequenced by Tsuchiya and his colleagues (10,11). The protein is deduced to be composed of 469 amino acids and have a molecular mass of 52,215 daltons (11). Hydropathy analysis predicts a short NH2 terminus, 11 transmembrane segments joined by short hydrophilic regions, and a long hydrophilic COOH terminus (13). Amino acid substitutions that alter sugar and/or cation specificity are predominantly found in four clusters on the periplasmic side of transmembrane segments 3, 6 , 9, and 10 (8,(12)(13)(14)(15)) and may define a common sugar/cation binding site when folded in the native conformation (13). The function, if any, of the long COOH tail has not previously been investigated.
This paper describes mutations of the rnelB gene that result in the truncation of the COOH tail at eight positions. Measurement of transport and cation coupling characteristics indicate that the hydrophilic domain of the tail has no direct role in substrate recognition or energy transduction but may play a role in stabilizing the carrier in the membrane.

Reagents-Melibiose
and methyl-P-D-thiogalactopyranoside (TMG)' were purchased from Sigma. [Meth~l-'~c]TMG and [3H] raffinose were from Du Pont-New England Nuclear. [3H]Melibiose was generously provided by Dr. Gerard Leblanc of the Departement de Biologie du Commissariat I'Energie Atomique, Villefranche-sur-Mer, France. Radiolabeled sugars were purified by paper chromatography on Whatman No. 3 MM chromatography paper using a mixed solvent phase of 3 parts of 1-propanol to 1 part of H20. cy -[36S]dATP (>600 mCi/mmol) was purchased from Amersham Corp. DNA sequencing reagents and restriction enzymes were purchased from New England Biolabs (Beverly, MA). All other chemicals were of reagent grade.
Construction of the melB Expression Plasmid pKKMB-EcoRI and Hind111 restriction sites were introduced into the melB DNA fragment of pSTY-91 (10) by oligonucleotide-directed site-specific mutagenesis as described below. The EcoRI site was introduced at nucleotide -46 GAAGGC-3' to 5'-GAATTC-3'. The Hind111 site was introduced at (numbering scheme of Ref. 11) by changing the native sequence 5'nucleotide 1473 by changing the sequence 5'-ATGCTT-3' to 5'-AAGCTT-3'. This produced a 1518-base pair EcoRI/HindIII DNA fragment containing the entire melB gene and Shine-Dalgarno sequence with little unessential DNA 3' or 5' of the structural gene. The fragment was ligated into the corresponding restriction sites of the expression vector pKK223-3 (Pharmacia LKB Biotechnology Inc.). The resulting plasmid, designated pKKMB, is schematically outlined in Fig. 1.
Site-specific Mutagenesis-Stop codons were introduced into the melB gene of pKKMB via oligonucleotide-directed site-specific mutagenesis according to the method of Eckstein (16, 17). Mutagenic oligonucleotides were synthesized using a Coder 300 DNA synthesizer (Du Pont Biotechnology Systems, Wilmington, DE) using standard phosphoramidite chemistry. Oligonucleotides were cleaved from the resin support according to the manufacturer's specifications, extracted three times with HzO-saturated 1-butanol, and used without further purification. The PstI/EcoRI melAB fragment of pSTY-91 (10) was ligated into M13mp18 and used to prepare single-stranded DNA template (18). EcoRI and Hind111 restriction sites were introduced by site-specific mutagenesis as described under "Materials and Methods." The resulting 1518-base pair EcoRIIHindIII DNA fragment containing the entire melB gene plus Shine-Dalgarno sequence was ligated into the corresponding restriction sites of the expression vector pKK223-3 (Pharmacia LKB Biotechnology Inc.). The resulting plasmid was designated pKKMB. "tac" designates the position of a trp-lac promoter (26). "rrnB TIT; designates the position of an rrnB TITZ ribosomal RNA transcription terminator (27,28 [35S]dATP. Elongation and chase reactions were carried out at 50 "C to eliminate the sequencing artifacts resulting from temperaturesensitive secondary structure characteristics of the DNA template. The entire melB gene and flanking regions were sequenced to ensure that only the desired mutation(s) had been incorporated. Areas of ambiguous interpretation were repeatedly sequenced until all ambiguities were resolved.
Uphill Sugar Transport-Transport was performed as described in the figure legends. Samples were rapidly filtered through a 0.65-pm pore size nitrocellulose membrane filter (Sartorious Filters Inc., Haywood, CA). Accumulated label was quantified by liquid scintillation transport by the pKKMB melibiose carrier. DW1 pKKMB was grown to early log phase at 37 "C in 1% Tryptone M63 containing 100 pg of ampicillin/ml and thiamine. Cells were washed twice and resuspended to 3 X lo9 cells/ml in M63 containing 10 mM NaCl (pH 7.2). The addition of 10 mM Na+ was necessary to facilitate TMG cotransport (4). Reactions were initiated by rapidly mixing 900 p1 of cells with 100 p1 of 1 mM stock sugar solutions containing 2 pCi/ml radioactive sugar. All reactions were at 25 "C. At the indicated times, 150-pl aliquots were rapidly filtered, washed with 5 ml of M63, and assayed as described under "Materials and Methods." Nonspecific adherence of label was determined by duplicate assays at 0 "C without incubation. Background values were subtracted and never exceeded 5% of total counts. Points represent the mean of duplicate samples.
-+ + -FIG. 3. Effect of temperature on the wild-type melibiose carrier. RVSmX containing pKK223-3 (parent vector without melB gene) were grown at 30 or 40 "C in 1% Tryptone M63 containing 100 pg of ampicillin/ml, thiamine, and 0 (-) or 10 mM (+) melibiose. TMG transport (solid bars) was determined as in Fig. 2, and initial rates are reported. a-Galactosidase activity (open bars) was determined using sonicated cells as described under "Materials and Methods." counting using Liquiscint (National Diagnostics, Somerville, NJ). a-Galactosidase Actiuity-a-Galactosidase activity was estimated by a colorimetric assay based on the hydrolysis of o-nitrophenyl agalactopyranoside using a modification of the method of Burstein and Kepes (20). Early log phase cells were washed twice and resuspended in M63 (100 mM potassium phosphate buffer, pH 7.2, 15 mM (NH&S04, 1 mM MgSO,) to a density of -5 X 10' cells/ml. Cells were sonicated to clarity with a probe sonicator and centrifuged for 60 min at 30,000 X g at 4 'C in a Sorvall SA-600 rotor to produce a clear supernatant containing the enzyme activity. a-Galactosidase activity was measured at 25 "C in a reaction mixture containing 3 mM o-nitrophenyl a-galactopyranoside, 3 mM MnS04, 100 mM pmercaptoethanol, 50 mM Tris-HC1 (pH 8.1), and 1 p~ NADf. Reactions were stopped by the addition of 250 mM Na2C03 and 40 mM EDTA and the absorbance measured at 420 nm. Enzyme activity was estimated by comparison to an o-nitrophenol standard curve.
pH Electrode Studies-Host strain DW1 (AmelAB AlacZY; Ref. 21) was used to measure H' movement by the truncated carriers. Strain RVSmX ( A b strAII, Ref. 22), which was used for all other physiological studies, could not be used for the measurement of H+ movement due to 3 small but significant number of lysed cells present following growth at 40 "C. The proteins released from these cells  The two amber stop codon truncations which removed 10 and 30 amino acids (pKKMB-ctlO and pKKMB-ct30, respectively) were not tested due to amber suppression by DW1. Therefore, only the ochre and opal stop codon truncations were assayed for proton movement. Cells were grown in 1% Tryptone medium 63 at 37 "C with vigorous shaking until early log phase (approximately four doublings) and washed 3 times in 120 mM choline chloride, 1 mM 0-mercaptoethanol (choline media). Cells were then resuspended in choline media containing 10 mM KSCN to -2 X 10'' cells/ml and made anaerobic by gently bubbling with N, for 40 min. Approximately 5 X 10" cells (2.5 ml of cell suspension) were used per recording. H' movement in response to the addition of melibiose (5 mM final concentration) was followed using a Radiometer (Copenhagen) GK231-C combined pH electrode. The stock melibiose solution was prepared in choline media and brought to pH 6.3 with acetic acid or KOH. Electrode response was calibrated following each assay by adding a known amount of anaerobic KOH. Sodium Electrode Studies-RVSmX containing pKKMB or a truncated derivative were grown in 1% Tryptone medium 63 at 37 "C with vigorous shaking until early log phase (approximately four doublings) 40 "C in 1% Tryptone M63 containing 100 pg of ampicillin/ml and thiamine. Cells were washed and TMG transport determined as described in Fig. 2. Points represent the mean of duplicate samples. and washed as for determination of H' movement. Cells were then resuspended in 100 mM Tricine buffer (pH 8.0 with tetramethylammonium hydroxide) containing 50 NM NaCl and 10 mM KSCN to -2 X 10" cells/ml and made anaerobic as above. Approximately 2 x 10" cells (10 ml of cell suspension) were used per recording. Na+ movement in response to the addition of melibiose (5 mM final concentration) was followed using a Radiometer (Copenhagen) G502 glass Na+specific and K401 calomel electrodes. A stock melibiose solution (500 mM) was prepared in Tricine buffer and made "Na+-free" by chromatography through Dowex 50%' (hydrogen form) pre-equilibrated with 2 volumes of 500 mM melibiose. Electrode response was calibrated after each assay by adding known quantities of anaerobic NaCl in Tricine buffer.
Radiolabeling of Cells and Preparation of Membranes-Cells of strain RVSmX containingpKKMB, pKKMB-ctl4, pKKMB-ct41, or pKKMB-ct56 were grown in 40 ml of 0.5% glycerol M63 containing 100 pg of ampicillin/ml and thiamine at 40 "C to early log phase. 3H-Labeled amino acids (125 pCi, Amersham mixture TRK.440) were added and growth continued for 2.5 doublings. Cultures of RVSmX containing pKK223-3 (parent plasmid Iacking the melB gene) were grown and labeled as above using "C-labeled amino acids (25 pCi, Amersham mixture CFB.104). Equal volumes of 3H-and "C-labeled cells were mixed and used to prepare cytoplasmic membranes as described by Osborn et al. (23). Membrane samples were dissolved in sample buffer (24) a t 37 "C for 30 min and electrophoresed through a 12.5% acrylamide SDS gel (24). The gel was fixed in methanokacetic acidwater (5010:40), stained with Coomassie Brilliant Blue, destained, and cut into l-mm sections. Radioactivity was extracted from the gel slices by treatment with 0.5 ml 90% Protosol (Du Pont-New England Nuclear), 10% Hz0 for 18 h at room temperature. Samples were mixed with 5 ml of Liquiscint, briefly vortexed, held overnight at room temperature to dissipate chemiluminescence, and radioactivity was quantitated by liquid scintillation spectroscopy. washed, and transport rates determined as described in Fig. 2. Points represent the mean of duplicate samples.

MelB Expression Plasmid pKKMB-A new
plasmid that expresses the temperature-resistant isoform of the melibiose carrier (4, 25) was constructed. The plasmid, designated pKKMB (Fig. l), contains the melB gene from pSTY91 (10) under inducible control of the strong tac promoter (26). Transcription of the melB RNA is terminated by the strong rrnB transcription terminator (27,28). This helps stabilize the host-vector system by inhibiting detrimental overexpression (29,30,31). When transformed into a ladQ background, melB expression is repressed unless induced by isopropyl-1-thio-@-D-galactopyranoside (data not shown). In a h I ' host such as DW1, expression of the carrier is only partially repressed. Uninduced DWl/pKKMB accumulates TMG 120-fold, melibiose 76-fold, and raffinose %-fold (Fig. 2). Unlike previous melB expression plasmids, the entire nucleotide sequence of pKKMB is known, and the insert contains less than 5% nonessential DNA.
The R VSmX Assay System-Measurement of plasmid-encoded melibiose carrier activity requires that the host strain be both lactose carrier and melibiose carrier negative. Two such strains of E. coli are available: DW1 and DW2 (21).
Unfortunately, these strains could not be used in the current study due to amber suppression by DW1 and DW2. Therefore transport characteristics of the truncated carriers were determined in RVSmX (Alac strAll, Ref. 22) grown at 40 "C by exploiting the heat lability of the chromosome-encoded melibiose carrier as discussed below. Two isoforms of the melibiose carrier that differ in temperature sensitivity are known (4,25). The wild-type carrier exhibits full activity when grown at 30 "C but is irreversibly Distribution of radioactivity in SDS-polyacrylamide gels of cytoplasmic membrane preparations. To determine if the melibiose carrier was present in the cytoplasmic membrane RVSmX containingpKKMB ( A ) , pKKMB-ctl4 ( E ) , pKKMB-ct41 (C), or pKKMB-ct56 ( D ) were grown in the presence of 3Hlabeled amino acids as described under "Materials and Methods" and mixed with an equal number of RVSmX containingpKK223-3 (vector without nelB gene) grown in the presence of "C-labeled amino acids. Cytoplasmic membranes were prepared from the pooled cells and electrophoresed through a 12% polyacrylamide SDS gel. The gel was fixed, cut into 1-mm slices, and 3H and ' .' C activity determined. Total counts for each isotope were normalized to 100% and a 3H:14C ratio calculated. The two profiles coincide except in the area of 3H excess corresponding to the melibiose carrier protein ( A and B ) . Approximate correspondence between gel slice and apparent molecular weight is indicated.
inactivated when grown at elevated temperatures; the temperature-resistant isoform is fully active throughout the temperature range that permits growth. The chromosomal melB of RVSmX is the temperature-sensitive isoform, while the melB used to construct pKKMB is the temperature-resistant isoform (10). Therefore, RVSmX can be used to assay melibiose carrier activity of pKKMB constructs if grown at 40 "C. This is illustrated in Fig. 3 which describes TMG transport and a-galactosidase activities of RVSmX pKK223-3 when grown at 30 and 40 "C. Very little melibiose transport activity attributable to the chromosomal melB gene product is detectable when grown at the elevated temperature, although agalactosidase activity is still present.
COOH-terminal Truncatiorw-"he COOH end of the melibiose carrier was truncated at eight locations by insertion of stop codons into the melB gene of pKKMB by site-specific mutagenesis. The location of the truncations and the nucleotide changes are listed in Table I. Thirty-six amino acids could be removed from the COOH terminus of the carrier without the complete loss of facilitated diffusion (Fig. 4) or uphill transport (Figs. 5 and 6). When 41 or more residues were removed, no transport of any type was detectable. To determine if this loss of transport activity was due to a direct perturbation of protein structure by the truncation or indirectly due to poor membrane insertion or post insertional proteolytic scavenging, we checked cytoplasmic membranes for the presence of the carrier protein using differential labeling of cells either expressing or not nonexpressing the melB gene (Fig. 7). In both cases (41 and 56 amino acid truncations) the carriers were absent from the cytoplasmic membrane. Wild-type and the 14-amino acid truncated carriers could be clearly detected by this technique.
Sugar specificity did not appear to be affected by truncation. Monosaccharide, disaccharide, and trisaccharide substrates (TMG, melibiose, and raffinose, respectively) were all transported. It is interesting to note that TMG showed significantly enhanced accumulation when 2, 10, or 14 amino acids were truncated from the carrier (Fig. 5). However, melibiose and raffinose accumulation remained comparable to that of the untruncated carrier (Fig. 6).
The ability of the truncated proteins to participate in H+coupled and Na+-coupled cotransport was measured using pH and Na+-specific electrodes (Fig. 8). All but one of the truncations that supported transport could use both cations. The sole exception, pKKMB-ct36 (36-amino acid truncation), displayed weak H+-coupled cotransport but no Na+-coupled cotransport. Given the poor sugar transport ability of the pKKMB-ct36 carrier in comparison to the other truncated carriers, it is possible that Na+-coupled cotransport was present but not detectable by the methods employed.

DISCUSSION
The results of the present study have shown that the COOH tail of the melibiose carrier plays no direct role in substrate recognition or energy transduction. When truncations were made at any site within the hydrophilic domain of the COOH terminus (Fig. 9), substrate specificity was unaffected, and the ability to accumulate substrate against concentration gradients remained qualitatively intact. 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 COOH-terminal portion of the protein (13).
Although significant transport by the truncated carriers could be demonstrated, the rate of transport was less than normal when 24, 30, or 36 amino acids were removed. This could be due to impairment of the translocation process at the level of the individual carrier or to the presence of fewer carriers within the membrane. Due to the lack of an antibody specific for the melibiose carrier, we were unable to distinguish between the possibilities. However, the counterflow data suggest that the decreased rates may be largely accounted for by a decrease in the number of carriers. As more of the COOH terminus was truncated, the initial rates and peak intracellular concentrations were progressively decreased and the time required to achieve peak intracellular concentration was progressively lengthened. Identical counterflow behavior was observed with the lactose carrier when the number of active transporters incorporated in the membrane was varied (32). This decrease in carrier number with progressive COOH truncation may be the result of progressively increased proteolytic scavenging of the membrane-inserted carriers, as discussed below.
When truncations were made within the hydrophobic domain of transmembrane helix ll (pKKMB-ct41 and pKKMB-ct56, Fig. 9), the carrier could no longer be found in the membrane. This suggests that the COOH 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. The recent finding that the 417-amino acid lactose carrier of E. coli can be truncated by as much as 366 amino acids (88% of the carrier being deleted) without impairment of insertion into minicell membranes (33) suggests that the truncated melibiose carriers were removed or degraded following initial insertion into the membrane. Lack of the hydrophilic tail to anchor helix 11 within the membrane or the failure of helix 11 to properly insert in the membrane in the absence of the anchor may target the protein for proteolytic removal. In contrast, truncations originating in the hydrophilic domain, all of which retained a COOH tail with at least four strongly hydrophilic residues (including a minimum of two charged residues), adequately anchored the terminal transmembrane segment and protected the carrier from degradation.