©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Reconstitution of the Purified Mannose Phosphotransferase System of Escherichia coli into Phospholipid Vesicles (*)

(Received for publication, August 30, 1994; and in revised form, December 12, 1994)

Qingcheng Mao (1) Thomas Schunk (2) Karin Flükiger (1) Bernhard Erni (1)(§)

From the  (1)Institute for Biochemistry, University of Bern, CH-3012 Bern Switzerland and the (2)Department of Biology, Philipps-University Marburg, D-3550 Marburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mannose transporter complex acts by a mechanism which couples translocation with phosphorylation of the substrate. It consists of a hydrophilic subunit (IIAB) and two transmembrane subunits (IIC, IID). The purified complex was reconstituted into phospholipid vesicles by octyl glucoside dilution. Glucose export was measured with proteoliposomes which were loaded with radiolabeled glucose and to which purified IIAB, cytoplasmic phosphorylcarrier proteins, and P-enolpyruvate were added from the outside. Vectorial transport was accompanied by stoichiometric phosphorylation of the transported sugar. Glucose added to the outside of the proteoliposomes was also phosphorylated rapidly but did not compete with vectorial export and phosphorylation of internal glucose. Glucose uptake was measured with proteoliposomes which were loaded with the cytoplasmic phosphoryl carrier proteins and P-enolpyruvate and to which glucose was added from the outside. Vectorial import and phosphorylation occurred with a higher specificity (K 30 ± 6 µM, k 401 ± 32 pmol of Glc/µg of IICD/min) than nonvectorial phosphorylation (K 201 ± 43 µM, k 975 ± 88 pmol of Glc/µg of IICD/min).

A new plasmid pTSHIC9 for the controlled overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme I, HPr, and IIA, and a simplified procedure for the purification of these proteins are also described.


INTRODUCTION

Uptake of hexoses and hexitols in bacteria is mediated by membrane protein complexes, the so-called enzymes II of the bacterial phosphotransferase system (PTS). (^1)They couple vectorial translocation with phosphorylation of the transported solute. Coupling is tight unlike in eukaryotic cells where transport and phosphorylation of glucose are mediated by a sequential pair of enzymes (transporter and kinase). However, phosphorylation of cytoplasmic substrates without transport, e.g. of glucose derived from maltose within the cell, has also been observed (Thompson and Chassy, 1985; Thompson et al., 1985; Nuoffer et al., 1988). There exist 16 enzymes II of different substrate specificity in Escherichia coli. They are composed of three functional units (IIA, IIB, IIC, or IICD; for nomenclature see Saier and Reizer(1992)) which occur either as protein subunits or domains of a multidomain protein. The IIC unit spans the membrane several times. It contains the substrate binding site. The IIA and IIB units are hydrophilic. They sequentially transfer phosphoryl groups from the ``high-energy'' phosphoryl carrier protein phospho-HPr to the substrate on the IIC subunit. Phospho-HPr in turn is regenerated by P-enolpyruvate in a reaction catalyzed by enzyme I. The collective of phosphoproteins which have additional functions in metabolite transport, chemotaxis, and metabolic regulation constitute the bacterial phosphotransferase system (for reviews, see Meadow et al.(1990), Erni(1992), and Postma et al.(1993)).

The mannose transporter of the PTS has a broad substrate specificity for mannose, glucose, and related hexoses. In addition, it acts as a host factor required for infection of E. coli by bacteriophage (Elliott and Arber, 1978). It consists of three subunits (Fig. 1). The IIC and IID subunits form a tight transmembrane complex which is sufficient for penetration of phage DNA (Erni et al., 1987). For transport and phosphorylation of sugars, the third subunit, IIAB, is also required. IIAB is reversibly associated with the IIC-IID complex (K 5-10 nM). (^2)It consists of two hydrophilic domains IIA and IIB which are linked through a hinge peptide-rich in Ala and Pro (Erni, 1989). IIA and IIB each contain one phosphorylation site (His and His) which relay the phosphoryl groups from HPr to the substrate on the IIC-IID complex. The isolated IIB domain together with the IIC-IID complex is sufficient for equilibrium phosphoryl exchange between Glc-6-P and Glc (Erni et al., 1989).


Figure 1: Hypothetical model of the mannose transporter and schematic representation of its action. A, vectorial transport and phosphorylation. Translocation and phosphorylation are tightly coupled. The transported solute (Glc) does not exchange with free solute (Glc). B, nonvectorial phosphorylation. Thin lines indicate the flow of phosphoryl groups from P-HPr via the IIA domain (dotted) and the IIB domain (diagonal hatching) to Glc. The two domains of IIAB are linked by an Ala-Pro-rich hinge. One IIC subunit (vertical hatching) and two IID subunits (horizontal hatching) form the transmembrane complex.



Due to the complexity of the system comprising two cytoplasmic phosphoryl carrier proteins (enzyme I and HPr) in addition to the transporter, sugar phosphorylation rather than vectorial transport activity is measured in routine assays (Kundig and Roseman, 1971). Of all the purified PTS transporters only the mannitol transporter could be reconstituted into phospholipid vesicles, so far (Elferink et al., 1990). The mannose transporter differs from the mannitol transporter in subunit composition and stoichiometry, chemistry of the active site, and protein structure. The molecular mechanism of vectorial solute transport and its coupling to phosphorylation are not understood. As a first step toward the elucidation of this process the mannose transporter was reconstituted into phospholipid vesicles and the vectorial transport and phosphorylation activity was measured. Also addressed was the question of substrate channeling (Srivastava and Bernhard, 1986; Srere, 1987; Welch and Easterby, 1994) between the transport and phosphorylation moieties, the hypotheses of transport regulation by the membrane potential (Reider et al., 1979; Robillard and Konings, 1981, 1982), and the proposition of a coupling of solute uptake with proton extrusion (Scarborough, 1985). To provide the soluble phosphoryl carrier proteins enzyme I and HPr, which are required in large amounts for the loading of proteoliposomes, a recombinant plasmid for their overexpression was constructed and methods for their purification are described.


MATERIALS AND METHODS

Bacterial Strains and Construction of Expression Plasmids

E. coli K-12 WA2127 is ptsGmanXYZ. WA2127DeltaHIC is ptsGmanXYZptsHptsIcrr. It was constructed by conjugative transfer into WA2127 of the ptsH ptsI crr deletion from strain UE7 (Hfr KL16 thi (ptsH ptsI crr) galR galP::Tn10; gift of W. Boos, University of Konstanz). Plasmid pTSPM8 encodes manY and manZ under the control of Ptac (Erni et al., 1987). The recombinant plasmid pTSHIC9 carries behind the Ptac promoter ptsH, ptsI, and crr encoding in this order HPr, enzyme I, and the IIA subunit of the glucose transporter. pTSHIC9 was constructed from the expression vector pJF119HE (Fürste et al., 1986) and plasmid pDIA3206 (contains 11 kilobase E. coli chromosomal DNA carrying ptsH ptsI crr; de Reuse and Danchin(1988)) as follows. (i) A 9.1-kilobase SalI-BamHI fragment containing ptsH ptsI crr from pDIA3206 was inserted into the polylinker of pJF119HE. (ii) Extra DNA at the 3` end of the ptsH ptsI crr coding region was deleted by BamHI and partial HpaI digestion of the plasmid followed by self-ligation of the desired 9.4-kilobase fragment. (iii) 5` noncoding chromosomal DNA was deleted unidirectionally starting from the polylinker toward the 5` end of ptsH (SalI and SphI digestion in the polylinker, unidirectional deletion with ExoIII nuclease for different time intervals, trimming with S1 nuclease, and self-ligation of the truncated fragments). (iv) Strain WA2127DeltaHIC was transformed with the self-ligated plasmids, plated on McConkey indicator plates (0.4% Glc, 100 µg/ml ampicillin), and colonies which weakly fermented Glc (complementation of IICB dependent Glc transport) were selected. (v) The selected colonies were replica plated on IPTG containing plates, and non-fermenting colonies were selected (strong overexpression of ptsH ptsI crr was found to inhibit Glc fermentation for unknown reasons). (vi) Cell lysates from IPTG-sensitive colonies were analyzed on polyacrylamide gels for overexpression of enzyme I, HPr, and IIA by Coomassie staining and autoradiography following incubation of the extracts with [P]P-enolpyruvate. The plasmid from a transformant displaying maximal overexpression was isolated and the noncoding region upstream of ptsH was sequenced by the dideoxy chain termination method using the Sequenase kit (U. S. Biochemical Corp.).

Overexpression and Purification of the IIC-IID Complex and of the IIAB Subunit

The IIC-IID complex was overexpressed in E. coli WA2127 transformed with plasmid pTSPM8 (Erni et al., 1987). Cells were grown at 37 °C to A = 1.5. Protein expression was induced with 50 µM IPTG and incubation continued for 5 h. The IIC-IID complex was solubilized with 2% octyl-beta-glucopyranoside (Sigma) and purified by isoelectric focusing in a U-tube in the presence of the same detergent (Erni et al., 1987). The IIC-IID complex from the peak fraction was concentrated in a Centricon 10 (Amicon) to 1 mg of protein/ml. The concentrated protein solution was used for reconstitution experiments. Protein concentrations were determined using the assay kit of Bio-Rad and bovine serum albumin as standard. The wild-type IIAB subunit was purified as described (Stolz et al., 1993).

Overexpression and Purification of Enzyme I, HPr, and IIA

Enzyme I and HPr were overexpressed in E. coli WA2127 transformed with plasmid pTSHIC9. Cells were grown at 37 °C to A = 1.5, protein expression was induced with 0.5 mM IPTG, and incubation continued overnight. Cells were collected by centrifugation, resuspended in a buffer A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM DTT, and 100 µg/ml phenylmethylsulfonyl fluoride), and ruptured by two passages through a French pressure cell (15,000 psi). Cell debris were sedimented by low speed centrifugation (10 min, 12,000 times g) and the membrane fraction by ultracentrifugation (90 min, 150,000 times g). Protamine sulfate was added to the high-speed supernatant to a final concentration of 0.33%. After removal of the precipitate by low speed centrifugation, enzyme I, IIA, and HPr account for 50% of protein in the clarified supernatant. The supernatant was applied to a DE-52 column (Whatman, 70-ml bed volume, equilibrated with buffer A), and eluted with a 0-700 mM NaCl gradient (buffer A, 2 ml/min flow rate). HPr eluted between 50 and 80 mM NaCl, IIA between 0.20 and 0.25 M NaCl, and enzyme I between 0.4 and 0.45 M NaCl. The HPr, IIA, and enzyme I containing fractions were pooled and concentrated by precipitation with 80% ammonium sulfate. The precipitates were dissolved in a small volume of buffer A and dialyzed against buffer A. IIA and HPr were further purified by gel filtration on Superdex 75 (Pharmacia Biotech Inc.), and enzyme I on Superdex 200. Protein concentrations were determined using a modified Lowry assay (Markwell et al., 1978) and bovine serum albumin as standard.

Reconstitution of the IIC-IID Complex

E. coliL-alpha-phosphatidylethanolamine (Type IX, Sigma) was dissolved in chloroform, dried in a round-bottom flask, and resuspended at a concentration of 20 mg/ml in reconstitution buffer (20 mM imidazole/HCl, pH 6.0, 150 mM KCl, 1 mM MgCl(2), 1 mM DTT). The suspension was homogenized by brief sonication in a bath-type sonicator and stored at -70 °C until use. 1 ml of phospholipid suspension (20 mg/ml) and 0.11 ml of 10% octyl-beta-D-glucopyranoside (Sigma) were added to a screw capped test tube, flushed with N(2), and vortexed for 30 s at room temperature. 0.05 ml of the isoelectrofocused IIC-IID complex (approximately 50 µg of protein) was added to the phospholipid/detergent solution. The solution was mixed gently, incubated for 1 h at 4 °C, pipetted into 30 ml of reconstitution buffer, mixed, and proteoliposomes were collected by centrifugation (1 h, 150,000 times g, 4 °C). The proteoliposomes were resuspended in 0.5 ml of phosphate buffer (50 mM KP(i), pH 7.5, 5 mM MgCl(2), 1 mM DTT, 40 mg/ml phospholipids, 100 µg/ml IIC-IID; molar lipid/protein ratio 30,000) and then stored at -70 °C until use.

Assay for Vectorial Import and Phosphorylation of Glc

The frozen proteoliposomes were thawed at room temperature. P-enolpyruvate and purified enzyme I, HPr, and IIAB were added to 250 µl of concentrated proteoliposomes to final concentrations of 4.5 mM, 0.3 µM, 2.5 µM, and 0.7 µM, respectively. The mixture was sonicated for 2 min at room temperature in a bath-type sonicator (TEC-40, TECSONIC, Switzerland). The sonicated proteoliposomes were frozen in liquid N(2) and thawed in a 22 °C water bath. Freezing and thawing was repeated 8 times. After sonication for 3 s in a bath-type sonicator, the proteoliposomes were purified by spin column gel filtration (Chonn et al.(1991). 1-ml tuberculin syringes plugged with glass wool were filled with Sephacryl S-300 (High Resolution, Pharmacia) equilibrated with buffer (50 mM KP(i), pH 7.5, 5 mM MgCl(2), 1 mM DTT) and hanging in 13 times 100-mm test tubes centrifuged for 2 min at 2000 rpm. Fills and centrifugations were repeated until the bed volume was 1 ml. A final spin of 5 min at 2000 rpm assured that the gel was uniformly packed and that excess buffer was removed. 80-µl aliquots of proteoliposomes were then applied and spun (1 min, at 1000 rpm). To drive the elution of proteoliposomes to completion, the column was washed with 80 µl of phosphate buffer. The proteoliposomes were diluted 4-fold into phosphate buffer to a final volume of 2 ml. The import reaction was started by adding [^14C]Glc (DuPont NEN, diluted to 10 mM, 4.5 mCi/mmol) to the desired concentration. Aliquots of 100 µl were withdrawn at the indicated time points and diluted into 2 ml of buffer (50 mM KP(i), pH 7.5, 5 mM MgCl(2), 1 mM DTT). To measure active import the diluted aliquots were immediately filtered through glass microfiber filters (GF/F, Whatman) under suction, the filters washed with 2 times 1 ml of phosphate buffer, and the radioactivity retained on the filters determined by liquid scintillation counting. To measure Glc phosphorylation, aliquots were diluted into 2 ml of 0.1% Triton X-100 and Glc-6-P was separated from Glc by anion-exchange chromatography as described (Erni et al., 1982). The kinetic constants were obtained by nonlinear fitting of the Michaelis-Menten equation to data points.

Assay for Vectorial Export and Phosphorylation, and Nonvectorial Phosphorylation of Glc

100 µl of D-[U-^14C]Glc (DuPont NEN, diluted to 10 mM, 4.5 mCi/mmol) was added to 250 µl of concentrated proteoliposomes. The mixture was sonicated and freeze-thawed as described above. To remove extravesicular [^14C]Glc the suspension was incubated for 15 min at 22 °C with hexokinase (4.5 mg/ml) and ATP (4.0 mM) and then spin column purified and 4-fold diluted as described above (5 mg/ml phospholipid, 0.18 µM IIC-IID). Enzyme I, HPr, and IIAB (final concentrations of 0.3, 2.5, and 0.7 µM) were added to the proteoliposomes and at 22 °C the transport reaction was started by adding P-enolpyruvate to a final concentration of 4.5 mM. Aliquots of 100 µl were withdrawn and [^14C]Glc retained in the proteoliposomes as well as [^14C]Glc-6-P formed were determined as described above. Proteoliposomes mock-loaded with Glc-free buffer were used to measure nonvectorial phosphorylation of external Glc. The reaction was started by adding enzyme I, HPr, IIAB, and P-enolpyruvate together with [U-^14C]Glc to the outside. Glc phosphorylation was measured by the ion-exchange method (Kundig and Roseman, 1971; Erni et al., 1982). To measure competition between vectorial export of internal [^14C]Glc and nonvectorial phosphorylation of external Glc, [C]Glc was added together with P-enolpyruvate. The kinetic constants were obtained by nonlinear fitting of the Michaelis-Menten equation to data points.

Generation of K Diffusion Potential

The proteoliposomes collected by ultracentrifugation were suspended in buffer A (150 mM KP(i), pH 7.5, 5 mM MgSO(4), 1 mM DTT) and loaded with [^14C]Glc as described. External K was removed together with external [^14C]Glc by spin column gel filtration through Sephacryl 300 equilibrated in buffer B (149 mM NaP(i), pH 7.5, 1 mM KP(i), 5 mM MgSO(4), 1 mM DTT). In this experiment, SO(4) was used to replace Cl in order to minimize the passive anion permeation. SO(4) has no effect on the phosphorylation activity of the mannose transporter (data not shown). The K diffusion potential was started by adding valinomycin (Sigma) to a final concentration of 40 nM. 5 min after the addition of valinomycin, the PTS-dependent extrusion of [^14C]Glc was started by adding P-enolpyruvate.

In parallel experiments the membrane potential formed by K-efflux from K-loaded proteoliposomes was measured with the fluorescence indicator 1,3,3,1`,3`,3`-hexamethylindoldicarbocyanine (NK529, Nippon Kankoh Shikiso Kenkyusho, Okayama, Japan) as described by Apell et al.(1985). 170 µl of K containing purified proteoliposomes (without [^14C]Glc) and 530 µl of buffer (149 mM NaP(i), pH 7.5, 1 mM KP(i)) containing 37 µM NK529 (added from 2.5 mM stock solution in 1:9 (v/v) ethanol/water) were mixed in a 1-ml cuvette. After the fluorescence signal reached a constant value (F(0)), valinomycin was added to a final concentration of 40 nM whereupon the fluorescence signal rapidly decreased to a new constant value (F). The K concentration was increased by small increments and the fluorescence change (DeltaF = F-F(0)) was monitored. There is a linear relationship between the membrane potential, which was calculated from the Nernst equation, and the fluorescence change: 0.64 F/mV. The fluorescence measurements were carried out with a Perkin-Elmer LS-5B luminescence spectrometer. The excitation wavelength was set to 620 nm (slit width 5 mm) and the emission wavelength to 680 nm (slit width 5 mm).

Protease Treatment of the Proteoliposomes

40 µl of spin column-purified proteoliposomes were incubated with the indicated amounts of trypsin (Sigma) or subtilisin (Serva) in potassium phosphate buffer (50 mM KP(i), 5 mM MgCl(2), 1 mM DTT, pH 7.5) with and without 2.7% Triton X-100 at room temperature in a final volume of 60 µl. Proteolysis was stopped by adding phenylmethylsulfonyl fluoride (3.5 mM, final concentration) at the indicated time intervals. Proteins were precipitated with 15% trichloroacetic acid, washed with acetone to remove detergent and phospholipid, and treated with formic acid/ethanol (1:2, v/v) to completely unfold the IIC subunit. The lyophilized protein samples were solubilized in 25 µl of sample buffer and separated by polyacrylamide gel electrophoresis. The proteins were stained with alkaline silver (Wray et al., 1981).


RESULTS

Overexpression and Purification of Enzyme I, HPr, and IIA

To maximize protein expression and minimize plasmid loss during fermentation, the pts operon encoding enzyme I, HPr, and IIA was put under the control of the Ptac promoter. The optimal distance between Ptac and the coding region was determined by progressively truncating the noncoding region upstream of ptsH and the screening of transformants for protein overexpression. Plasmid pTSHIC9 (Fig. 2A) supporting the strongest overexpression was isolated and the 5` region between ptsH and Ptac was sequenced. It comprises 148 noncoding nucleotides. It ends 16 nucleotides downstream of the -10 region of the pts P1 promoter (de Reuse et al., 1992). Enzyme I is the most prominent of all E. coli proteins in whole cell lysates, and IIA and HPr also appear as prominent bands on a Coomassie-stained electropherogram (Fig. 2B, lane 1). The proteins were purified by procedures adapted from the Roseman laboratory (Kukuruzinska et al., 1982; Nakazawa and Weigel, 1982; Meadow, 1982). After only two steps of purification the three proteins appear homogeneous as judged by polyacrylamide gel elecrophoresis (Fig. 2B), and 90% of the PTS activity present in the cytoplasmic fraction is recovered (Schunk, 1992). However, the IIA preparation was cross-contaminated with HPr activity as indicated by the strong background of PTS activity of an assay mixture from which exogenous HPr was omitted. This residual HPr activity could be completely removed by two extra steps of IIA purification: anion exchange chromatography on Mono-Q (Pharmacia) and gel filtration on Superdex 75. No mutual cross-contamination of enzyme I and HPr and no contamination of these proteins by IIA could be detected using elevated background activity in the sugar phosphotransferase assay as the criterium.


Figure 2: Plasmid map of pTSHIC9 (A) and Coomassie-stained polyacrylamide gels of enzyme I, HPr, and IIA (B). Lane 1, whole cell lysate from IPTG-induced cells; lane 2, purified HPr; lane 3, purified enzyme I; lane 4, purified IIA (the double band represents full-length and NH(2) terminally processed forms of IIA (Meadow et al., 1986)). The molecular mass markers are phosphorylase b (94 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and alpha-lactalbumin (14.4 kDa). The 8-25% polyacrylamide gel was prepared according to Fling and Gregerson (1986).



Preparation and Physical Characterization of Proteoliposomes

Proteoliposomes were formed by dilution of the purified, beta-octyl glucoside solubilized IIC-IID complex into a detergent-free buffer containing a 30,000-fold molar excess of E. coli phospholipids. 80-90% of the PTS activity present in the IIC-IID preparation could be recovered in the sedimented proteoliposome preparation. Detergent dialysis also afforded proteoliposomes but their IIC-IID activity was below 20%. The uranyl acetate-stained vesicles had diameters of between 200 and 500 nm (results not shown). The orientation of IIC-IID in the membrane is random as concluded from proteolysis experiments. Treatment of proteoliposomes with subtilisin resulted in a 50% conversion of the 29-kDa IID subunit into a new 28-kDa fragment (Fig. 3A). Trypsin removed 50% of the IID (Fig. 3B) from the intact vesicles. No tryptic fragments of IID could be detected by alkaline silver stain (Fig. 3B) and immune blots (results not shown). When the proteoliposomes were solubilized with detergent prior to proteolysis, conversion of the IID subunit was complete (Fig. 3). The IIC subunit behaved differently. It was cleaved into smaller fragments by subtilisin and trypsin, but cleavage did not go to completion after detergent solubilization of the proteoliposomes (Fig. 3). The reason for this behavior is not clear. After detergent solubilization, 50% of (nonvectorial) phosphotransferase activity could be recovered from the trypsin-treated proteoliposomes. Only 20% activity could be recovered from the subtilisin-treated proteoliposomes, suggesting that the IIC-IID complex is accessible to subtilisin in both orientations. This results in more than 50% inactivation but cleavage from only one side afforded a product of different electrophoretic mobility.


Figure 3: Proteolysis of the IIC/IID complex in proteoliposomes. A, 50% of the IID (D) subunits are processed to an intermediate (D*) upon incubation of intact proteoliposomes with subtilisin (S), close to 100% after solubilization of proteoliposomes with Triton X-100. Digestion of IIC (C) to C* is not complete. B, 50% of the IID (D) subunits are processed upon incubation of intact proteoliposomes with trypsin (T). No distinct IID intermediate is formed and digestion of IIC (C) to C* is not complete. The 20% polyacrylamide gels were stained with alkaline silver.



Transport and Phosphorylation Activity

The IIC-IID complex is oriented randomly in the proteoliposomes. However, by adding the soluble phosphoryl carrier proteins and P-enolpyruvate to only one of the two compartments a unique direction of transport can be imposed upon the reconstituted system. Uptake can be measured if the proteoliposomes are loaded with the phosphoryl carrier proteins and [^14C]Glc is added to the outside (``right-side out''), efflux if they are loaded with [^14C]Glc and the soluble phosphotransferase proteins are added to the outside (``inside-out''). The inside-out orientation allows for manipulation of protein-protein interactions occurring at the cytoplasmic face of the membrane but is of limited use for kinetic studies because intravesicular glucose is quickly depleted. The right-side out orientation allows assay for time-dependent solute accumulation but intravesicular conditions are not easily manipulated.

Import of [^14C]Glc was measured with proteoliposomes which were loaded with purified soluble PTS proteins and P-enolpyruvate. [^14C]Glc was added to start the uptake reaction (Fig. 4). 80% of the glucose originally present at a concentration of 5 µM was concentrated in the proteoliposomes and accumulation was accompanied by phosphorylation (Fig. 4A). The k and K(m) of vectorial transport by proteoliposomes were 401 ± 32 pmol of Glc/µg of IIC-IIDD/min and 30 ± 6 µM, respectively (Fig. 4, B and C). Assuming that the molecular mass of the functional unit of the mannose transporter is 90 kDa and that only 50% are correctly oriented and therefore active, a turnover number of 1.2 s can be calculated. No uptake of [^14C]Glc could be detected, when P-enolpyruvate, enzyme I, HPr, IIAB, and [^14C]Glc were added to the outside of the proteoliposomes (results not shown), indicating that transport occurs only if the solute ([^14C]Glc) and the phosphoryl transfer components are on opposite sides of the membrane.


Figure 4: Uptake and phosphorylation of glucose. A and B, IIC-IID containing proteoliposomes are loaded with IIAB, the phosphoryl carrier proteins, and P-enolpyruvate. The uptake reaction is started (arrow) by addition of [^14C]Glc to the outside. Open symbols, uptake; closed symbols, phosphorylation. A background of 5 pmol of Glc has been subtracted. A, 0.1 µM IIC-IID, IIC-IID:phospholipid ratio = 1:30,000, 5 µM Glc (box, ), 50 µM Glc (circle, bullet). B, 0.04 µM IIC-IID, IIC-IID:phospholipid ratio 1:75,000, 3.8 µM (circle), 7.5 µM (), 15 µM (), 28 µM (up triangle), 56 µM (box), and 112 µM Glc (down triangle). C, Lineweaver-Burk plot of initial rates of uptake activity from B. k = 1.2 ± 0.1 s and K = 30 ± 6 µM. Shown are means and S.D. of three independent transport experiments done with a single batch of reconstituted proteoliposomes.



Observations with intact bacteria indicated that PTS transporters not only catalyze vectorial transport with phosphorylation but also phosphorylation of substrates in the cytoplasm (nonvectorial phosphorylation, e.g. of glucose generated in the cell from maltose). When [^14C]Glc is added together with the soluble phosphoryl carrier proteins to the outside of IIC-IID containing proteoliposomes, Glc-6-P is formed rapidly without concommitant transport (Fig. 5A). The k and K(m) of nonvectorial phosphorylation by proteoliposomes are 975 ± 88 pmol of Glc-6-P/µg of IIC-IID/min and 201 ± 43 µM, respectively (Fig. 5B). Phosphorylation without transport is therefore 2-3 times faster than vectorial phosphorylation.


Figure 5: Nonvectorial phosphorylation of glucose. A, P-enolpyruvate and the phosphoryl carrier proteins are added to the outside of IIC-IID containing proteoliposomes (0.1 µM IIC-IID, IIC-IID:phospholipid ratio 1:30,000). The reaction was started by addition of [^14C]Glc to 15.1 µM (circle), 38.5 µM (down triangle), 76.3 µM (box), 151.0 µM (up triangle), 298.5 µM (), and 579.0 µM () final concentration. B, Lineweaver-Burk plot of initial rates of phosphorylation activity from A. k = 2.9 ± 0.3 s and K = 201 ± 43 µM. Shown are means and S.D. of three independent experiments done with a single batch of reconstituted proteoliposomes.



This nonvectorial phosphorylation raises two questions. First, passive diffusion of solute followed by nonvectorial phosphorylation could in principle be misinterpreted as true vectorial phosphorylation in the reconstituted system. Second, transport coupled to phosphorylation and nonvectorial phosphorylation could be competing reactions. The two problems were addressed by measuring the efflux of [^14C]Glc in the presence and absence of nonlabeled external glucose.

To test for passive and IIC-IID-mediated facilitated diffusion, (proteo)liposomes were loaded with [^14C]Glc and purified by spin column chromatography (without the hexokinase treatment described under ``Materials and Methods''). By adding hexokinase and ATP, a constant amount of Glc could be converted to Glc-6-P in the external compartment of pure liposomes as well as of proteoliposomes containing the IIC-IID complex. This amount did not change when the soluble phosphoryl carrier proteins were added as long as either P-enolpyruvate or the IIAB subunit were omitted (Fig. 6). The amount of Glc-6-P did, however, increase immediately above this background when P-enolpyruvate (Fig. 7B) was added, and this increase was accompanied by a stoichiometric decrease of trapped glucose (Fig. 7A). This result indicates that (i) export of [^14C]Glc concomitant with phosphorylation is catalyzed only by the complete phosphotransferase system, (ii) passive and IIC-IID-dependent facilitated diffusion of [^14C]Glc do not occur on the time scale of up to 1 h, and (iii) a small amount of [^14C]Glc which was not removed by spin column purification alone remains accessible from the outside. Henceforth, residual nontrapped glucose was removed with hexokinase prior to spin column purification as described under ``Materials and Methods.'' When IIAB, enzyme I, HPr, and P-enolpyruvate were added to the hexokinase-treated and spin column-purified proteoliposomes, rapid export of trapped [^14C]Glc concomitant with phosphorylation could be measured (Fig. 7). Export of glucose and phosphorylation are strictly coupled in a 1:1 molar ratio.


Figure 6: Nonspecific carry over (adsorption) and passive diffusion of Glc out of proteoliposomes. Proteoliposomes were loaded with [^14C]Glc and purified by spin column centrifugation (time 0). Hexokinase (1 mg/ml) and ATP (4.0 mM) were added after 20 min (arrow) and the formation of Glc-6-P was monitored by ion exchange chromatography. Pure liposomes without IIC-IID (bullet); proteoliposomes with IIAB, enzyme I, HPr, but without P-enolpyruvate () proteoliposomes with enzyme I, HPr, P-enolpyruvate but without IIAB (). A constant amount of Glc-6-P is formed from external glucose that does not further increase with time.




Figure 7: Export and phosphorylation of glucose. IIC-IID containing proteoliposomes (0.1 µM IIC-IID, IIC-IID:phospholipid ratio 1:30,000) are loaded with [^14C]Glc (2.85 mM). The export reaction is started (arrow) by addition to the outside of IIAB, HPr, enzyme I, and P-enolpyruvate without (circle, bullet) and with (down triangle, ) 17.5 mM unlabeled Glc. A, export of trapped [^14C]Glc. B, vectorial phosphorylation, formation of Glc-6-P. External glucose does not compete with export and phosphorylation of [^14C]Glc (compare triangles and circles). box, diffusion of [^14C]Glc observed when P-enolpyruvate is omitted from the incubation mixture. Shown are means and S.D. of three independent experiments done with a single batch of reconstituted proteoliposomes.



Are nonvectorial phosphorylation, as described above (Fig. 5) and vectorial transport and/or phosphorylation of transported substrate (Fig. 7) competing reactions? To address this question export and phosphorylation of trapped [^14C]Glc was measured in the presences of increasing amounts of external [C]Glc. As shown in Fig. 7, a 6-fold molar excess of external [C]Glc neither slows down the rate of [^14C]Glc export (Fig. 7A) nor its phosphorylation (Fig. 7B). This indicates that (i) nonvectorial phosphorylation and vectorial transport are not competing reactions and (ii) that [^14C]Glc does not equilibrate with [C]Glc between the translocation and phosphorylation reaction and coupling between these two steps therefore must be tight (Fig. 1). No difference was observed whether [C]Glc was added simultaneously with the cytoplasmic phosphoryl carrier proteins (Fig. 7) or whether it was added before (results not shown). Reconstituted IIC-IID does catalyze exchange equilibration between internal and external glucose.

Phosphotransferase Activity and Membrane Potential

It has been proposed that the activity of PTS transporters is regulated by the transmembrane electrochemical potential (Robillard and Konings, 1981, 1982). The rate and extent of solute accumulation by intact cells increased when the proton electrochemical gradient was abolished by uncouplers or respiratory chain inhibitors (Reider et al., 1979; Singh et al., 1985; Nuoffer et al., 1988). To rationalize this inverse correlation between membrane potential and transport activity, a mechanism involving the release of a proton on the periplasmic side concomitant with sugar phosphorylation was suggested (Scarborough, 1985). Reconstitution of the purified mannose transporter allowed testing for a possibly direct effect of the membrane potential on the transport activity. The extrusion from and the import of [^14C]Glc into K-loaded proteoliposomes was measured in the absence and in the presence of valinomycin. The calculated K diffusion potential was 120 mV, positive outside. According to the predictions the transport activity should have increased in the extrusion experiment (negative polarization with respect to the direction of transport, inside-out orientation) and decreased in the uptake experiment (positive polarization, right-side out orientation). However, in neither case could a difference be detected (results not shown). Similarly the addition of the uncoupler dinitrophenol had no effect on extrusion or uptake of [^14C]Glc.


DISCUSSION

The purified mannose transporter of the bacterial phosphotransferase system was reconstituted by detergent dilution into proteoliposomes and two activities, vectorial transport concomitant with phosphorylation and nonvectorial phosphorylation were measured. Transport is coupled to phosphorylation in a 1:1 ratio. The K(m) of the transport reaction is 30 ± 6 µM. A turnover number of 1.2 ± 0.1 s can be calculated, assuming that the minimal functional unit consists of one IIC and two IID subunits (Rhiel et al., 1994), and that at most 50% of the complexes are correctly oriented and active (probably an overestimate). The turnover number of the PTS transporter for mannitol is 4 s (Elferink et al., 1990) and similar numbers can be calculated from reported experiments with the purified H/lactose transporter (3.5 s, Newman et al.(1981); 0.6 s, Consler et al.(1993)), the intestinal Na/glucose transporter (4 s, Peerce and Clarke(1990)), and the histidine transporter (1.1 min, Bishop et al.(1989)). Nonvectorial phosphorylation has an approximately 7-fold higher K(m) and a 3-fold faster k than the transport reaction. It is likely that solute translocation requires more extensive protein motion than the binding and release reaction during nonvectorial phosphorylation, and that the former reaction therefore is slower than the latter. A similar difference of catalytic constants was also observed with the mannitol transporter (Elferink et al., 1990).

Although nonvectorial phosphorylation on the one hand and phosphorylation coupled with solute translocation on the other hand are mediated by the same protein complex, no cross-inhibition between the two reactions could be observed. Neither the export of [^14C]Glc out of proteoliposomes nor phosphorylation of the exported solute could be inhibited by Glc added to the outside of the proteoliposomes. Noncompetition between nonvectorial and vectorial phosphorylation indicates that the transported glucose is not in diffusion equilibrium with glucose in the bulk phase. Transport and phosphorylation are either mechanistically coupled or the rate of phosphorylation of bound glucose is much faster than the rate by which unphosphorylated glucose could dissociate (dissociation being a precondition for exchange; Fig. 1). This is a clear example of tight channeling by a multifunctional enzyme which catalyzes two sequential reactions (Srivastava and Bernhard, 1986; Srere, 1987). No competition between translocation and nonvectorial phosphorylation could be detected within the experimental error of the export assay. However, the initial rate of export could not be measured accurately because export is fast and due to the small intravesicular volume of very short duration. Small differences might therefore have gone unnoticed.

The results summarized above differ in three respects from recent observations with the mannitol transporter which indicated facilitated diffusion, exchange, and uncoupling of transport and phosphorylation. (i) Purified IICBA reconstituted into proteoliposomes mediated facilitated diffusion in a way which was saturable and specific for mannitol (Elferink et al., 1990). (ii) Addition of unlabeled Mtl to the exterior of inside-out membrane vesicles resulted in the complete exchange of [^3H]Mtl bound to the interior (Lolkema et al., 1991). (iii) Over 50% of the radiolabeled mannitol bound to the periplasmic side of IICBA containing inside-out oriented membrane vesicles exchanged with unlabeled mannitol before it became phosphorylated, indicating that mannitol dissociates after translocation at a rate comparable to that of phosphorylation. The differences could have several reasons. (i) The two transporters are very different with respect to amino acid sequence, active site residues (histidine versus cysteine in the second phosphorylation site; Pas and Robillard(1988), Erni et al.(1989), and Pas et al.(1991)), and subunit composition (4 domains in 3 subunits versus 3 domains in 1 subunit) and the molecular mechanism of their action might be correspondingly different. (ii) Facilitated diffusion catalyzed by the IIC-IID complex went unnoticed because it was indistinguishable from passive diffusion of L-glucose and diffusion of D-glucose out of protein-free liposomes (results not shown).

The proteolysis data are not easily reconciled with the proposition of Beneski et al.(1982) that the II complex is symmetrically oriented in the membrane. They observed that II interacts with 2-deoxyglucose and phosphorylate this sugar when the phosphoryl carrier proteins are located either inside of right-side out membrane vesicles (resulting in uptake and phosphorylation) or outside the vesicles (nonvectorial phosphorylation). If the two surfaces exposed toward the aqueous phases were identical, proteolysis should proceed to 100% and not only 50% as observed (Fig. 3). However, functional symmetry found by Beneski et al.(1982) does not necessitate a structural symmetry of the complex. Both faces of an asymmetrical complex could in principle have independent sugar phosphorylation activity, and noncompetition between vectorial transport and nonvectorial phosphorylation could then be explained. However, it appears more likely to us that some randomization of sidedness might have occurred during preparation of membrane vesicles by Beneski et al.(1982).

The present reconstitution method is useful whenever nonvectorial phosphorylation and vectorial transport must be investigated in parallel and when interference with PTS activity by other membrane components has to be excluded. It will be used as an assay complementary to in vivo uptake studies and in vitro measurement of nonvectorial phosphorylation.


FOOTNOTES

*
This work was supported by Grants 31-29795.90 from the Swiss National Science Foundation and the Deutsche Forschungsgemeinschaft (Er 147/1-1), and by contributions from the Sandoz-Stiftung, Basel, and the Central Laboratories of the Swiss Red Cross, Bern. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Biochemistry, University of Berne, Freiestr. 3, CH-3012 Bern, Switzerland. Tel.: 41-31-6314346; Fax: 41-31-6314887; erni{at}ibc.unibe.ch.

(^1)
The abbreviations used are: PTS, P-enolpyruvate-sugar phosphotransferase system; IIC and IID, transmembrane subunits of the mannose transporter; IIAB, hydrophilic subunit of the mannose transporter; IICBA, mannitol transporter; HPr, histidine-containing phosphocarrier protein of the PTS; IICB, transmembrane subunit of the glucose transporter; IIA, cytoplasmic subunit of the glucose transporter; ptsH ptsI crr, genes coding for HPr, enzyme I and IIA; ptsG, gene encoding IICB; IPTG, isopropyl-beta-D-thiogalactopyranoside; DTT, dithiothreitol.

(^2)
B. Erni, unpublished data.


ACKNOWLEDGEMENTS

We thank R. Lanz for expert technical assistance, Dr. C. Nuoffer for the construction of plasmid pTSHIC9, Drs. H. de Reuse and A. Danchin for plasmid pDIA3206, Prof. W. Boos for strain UE7, and Prof. H. Oetliker for assistance in measuring of the membrane potential. Electron microscopy was performed by T. Wyler.


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