©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A String of Enzymes, Purification and Characterization of a Fusion Protein Comprising the Four Subunits of the Glucose Phosphotransferase System of Escherichia coli(*)

(Received for publication, May 3, 1995)

Qingcheng Mao (1) Thomas Schunk (2) Basil Gerber (1) Bernhard Erni (1)(§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A multidomain protein comprising the four subunits of the glucose phosphotransferase system of Escherichia coli was constructed by fusion of the transmembrane subunit IICB and the three cytoplasmic proteins, IIA, HPr, and enzyme I. The subunits were linked in the above order with Ala-Pro-rich linkers; the fusion protein was overexpressed in E. coli and purified by Ni chelate affinity chromatography. Approximately 3 mg of the fusion protein could be purified from 1 liter of culture. The phosphotransferase activity of the purified fusion protein was 3-4 times higher than that of an equimolar mixture of the isolated subunits. The mannose transporter, which also requires enzyme I and HPr, was not an effective competitor in the overall phosphoryltransfer reaction when the fusion protein was used, whereas it was a competitor when an equimolar mixture of the separate subunits was employed. Transphosphorylation activity of the fusion protein was almost indistinguishable from the wild-type IICB. Addition of extra IICB subunit could significantly stimulate the phosphotransferase activity of the fusion protein, addition of extra IIA subunit and enzyme I, in contrast, was slightly inhibitory, and HPr had almost no effect. An optimal detergent-lipid ratio is required for maximum activity of the fusion protein. Our results suggest that Ala-Pro-rich linker sequences may be of general use for the construction of catalytically active fusion proteins with novel properties.


INTRODUCTION

The enzymes of a metabolic pathway can interact with each other to form highly organized enzyme complexes (Reed, 1974; Perham, 1975; Srivastava and Bernhard, 1986a, 1986b; Srere, 1987; Perham, 1991). These interactions range from weak and transient to covalent as found in multifunctional enzymes (Srere, 1985, 1987). For the enzymes in the tricarboxylic acid cycle, interactions have been demonstrated between six and eight sequential enzymes, and all of the enzymes have been found to bind to the inner surface of the mitochondrial inner membrane (D'Souza and Srere, 1983; Robinson and Srere, 1985; Datta et al., 1985; Brent and Srere, 1987). Five of the tricarboxylic acid cycle enzymes of Escherichia coli can also be isolated as a high molecular weight complex (Barnes and Weitzman, 1986). There are several possible reasons for such organization, including enhancement of catalytic activity and substrate channeling. A complex of functionally related enzymes might be more efficient in catalyzing a multistep metabolic sequence than would be a collection of independent enzymes. In other words, complex formation provides a means of concentrating catalysts rather than having them randomly distributed in the cell. Complex formation also offers a way of segregation of enzymes that would otherwise compete for the same intermediate. Such metabolic channeling has been invoked frequently in speculations on the role of multienzyme complexes in cellular economy, and has been suggested to be important in many cases, for instance, in shortening the transient times of overall reactions in metabolic processes (Welch and Easterby, 1994).

The bacterial phosphotransferase system (PTS) (^1)comprises multienzyme complexes that are composed of membrane-embedded subunits and cytoplasmic proteins. The system as a whole has at least three functions, uptake of carbohydrates coupled to their phosphorylation, regulation of metabolic pathways (catabolite repression, inducer exclusion), and chemotactic signaling (for reviews see Meadow et al.(1990) and Postma et al.(1993)). The glucose-specific phosphotransferase system of E. coli in particular consists of the membrane-embedded IICB subunit and of the three soluble subunits, IIA, HPr, and enzyme I. The four subunits form a phosphorylation cascade, which in the order P-enolpyruvate enzyme I HPr IIA IICB Glc transfers phosphorylgroups from P-enolpyruvate to the transported sugar (Glc). Enzyme I is a homodimeric protein (subunit M(r) 63,489) of as yet unknown structure. The enzyme I monomer consists of two equally sized domains (Neyroz et al., 1987; Han et al., 1990; LiCalsi et al., 1991). The carboxyl-terminal domain catalyzes phosphoryltransfer from phosphoenolpyruvate to His located on the amino-terminal domain (Alpert et al., 1985; Saffen et al., 1987). HPr is a two-layer alpha/beta protein (M(r) 9109), which is phosphorylated on His in the loop between beta1 and alpha1 (Jia et al., 1994). Amino and carboxyl termini are at the same edge but on opposing ends of the beta-sheet with the carboxyl terminus close in space to the active site histidine. IIA (M(r) 18,556) is a beta-sheet sandwich with six antiparallel strands on either side (Worthylake et al., 1991). The phosphorylatable His is located in the center of a shallow depression. The amino and carboxyl termini of this monomeric subunit are close in space. They are near the amino and carboxyl ends of the two terminal beta-strands, which are direct neighbors in an antiparallel beta-sheet. Besides serving as a phosphorylcarrier between HPr and IICB, IIA has regulatory functions. Unphosphorylated IIA binds and inhibits other transporters such as the lactose carrier and enzymes participating in the carbohydrate metabolism such as glycerol kinase (Postma et al., 1993; Hurley et al., 1993; Saier and Reizer, 1994). Phosphorylated IIA activates adenylcyclase by an unknown mechanism. IICB is a homodimeric protein (subunit M(r) 50,647), which catalyzes vectorial translocation and phosphorylation of glucose. The IICB monomer consists of two domains. The amino-terminal IIC domain (residues 1-386) is predicted to span the membrane 8 times (Buhr and Erni, 1993). It contains the sugar binding site. The carboxyl-terminal IIB domain (residues 391-477) is a two layer alpha/beta protein (Golic Grdadolnik et al., 1994). (^2)It becomes phosphorylated on Cys in the loop between beta1 and beta2 (Meins et al., 1993). The IIC and IIB domains can be expressed as independent protein subunits that in combination retain catalytic activity (Buhr et al., 1994) or can be circularly permuted, affording a ``IIBC'' protein with catalytic activity comparable to wild-type IICB. (^3)This indicates that the amino and carboxyl termini of IICB both are on the cytoplasmic face of the membrane and possibly close in space.

The sequential phosphoryltransfer between the different proteins necessitates protein-protein interactions. It has been suggested that the components of the PTS exist as membrane-associated multiprotein complexes rather than as individual components (Scholte et al., 1982; Brouwer et al., 1982; Saier et al., 1982; Misset et al., 1983). The actual composition of the complexes might vary, depending on the metabolic requirements of the cell. Steady-state kinetic studies with PTS proteins of E. coli have revealed a K of 5 µM for the interaction between enzyme I and HPr and of 0.3 µM for the interaction of phospho-HPr with IIA. In contrast, the K of IICB for IIA exceeds 20 µM (Reizer et al., 1992; Postma et al., 1993). Autophosphorylation of enzyme I with phosphoenolpyruvate is enhanced strongly in the presence of HPr, indicating a stabilizing and cooperative interaction between phospho-HPr and phosphoenzyme I (Erni, 1986). Similarly, IIA increases the rate of phosphoryltransfer from phospho-IICB to glucose and slows down spontaneous hydrolysis of phospho-IICB (Erni, 1986). However, the evidence for membrane-associated complexes is circumstantial and they could not be isolated.

Fusion of sequential enzymes by molecular biological techniques has been proposed as a means for the study of proximity effects of complexes that tend to dissociate due to dilution during isolation (Blow and Mosbach, 1991). A number of bifunctional fusion proteins that catalyze sequential reactions have been constructed (Blow, 1987; Ljungcrantz et al., 1989; Lindbladh et al., 1994). The kinetic benefits of such fusions were shorter transient times, and an apparent sequestering of the intermediates by the fusion protein when compared with the system of free enzymes. Examples of successful fusions are the fusions of yeast mitochondrial citrate synthase and malate dehydrogenase (Lindbladh et al., 1994), the fusion of subunits I, II and III of the cytochrome b0 ubiquinol oxidase of E. coli (Ma et al., 1993), and the fusions between cytochrome P450 and the flavoprotein domain of the NADPH P450 reductase of homologous as well as heterologous origin (Fisher et al., 1992; Shet et al., 1994).

Here we characterize a fusion protein containing the four protein subunits IICB, IIA, HPr, and enzyme I of the glucose-specific phosphotransferase system. To facilitate the domain movement required for active site coupling and optimal catalytic activity, a naturally occurring linker peptide was used to connect the subunits. Numerous oligopeptide sequences linking domains in protein tertiary structures have been described (Erni, 1989; Argos, 1990; Perham, 1991). From these, the Ala-Pro linker was chosen that occurs in the decarboxylases and dehydrogenases, where it links the biotinylated and lipoylated domains, respectively (Perham, 1991) and in proteins of the bacterial phophotransferase system (Erni et al., 1987; Peri and Waygood, 1988; Wu et al., 1990).


EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Growth Media

Strain ZSC112LDeltaHIC is glk ptsG manZ ptsH ptsI crr. It was constructed by conjugative transfer into ZSC112L (Curtis and Epstein, 1975) of the ptsH ptsI crr deletion from strain UE7 (Hfr KL16 thi Delta(ptsH ptsI crr) galR galP::Tn10; a gift of W. Boos, University of Konstanz, Konstanz, Germany). The expression vector pJF119EH contains lacI and Ptac (Frste et al., 1986), pTSG11 encodes IICB (Buhr et al., 1994) under the control of the Ptac promoter, and pTSHIC9 encodes HPr, enzyme I, and IIA also under the control of the Ptac promoter (Mao et al., 1995). Cells were grown at 37 °C in LB medium containing 0.1 mg/ml ampicillin.

Plasmid Construction

The plasmid pJFGCHI was assembled stepwise using the expression vector pJF119EH and coding regions taken from plasmids pTSG11 and pTSHIC9. One DNA duplex encoding the Ala-Pro-rich linker I (see Fig. 1B) was taken from plasmid pTSL23 (Erni et al., 1987). The other duplex encoding linker II (Fig. 1B) from the Rhodobacter capsulatus multiphosphoryl transfer protein (Wu et al., 1990) was chemically synthesized. Gapped duplex mutagenesis (Stanssens et al., 1989) and polymerase chain reaction site-directed mutagenesis (Mikaelian and Sergeant, 1992) were used to introduce compatible restriction sites at the ends of the sequences to be assembled. Standard procedures were used for ligation, transformation, restriction analysis, and plasmid preparation (Sambrook et al., 1989). Double-stranded plasmid DNA was sequenced by the dideoxynucleotide chain termination method using the Sequenase® kit (U. S. Biochemical Corp.). Intermediates in the stepwise assembly of pJFGCHI were the plasmids pJFGC encoding a fusion protein between IICB and IIA with linker I, pJFCH encoding a fusion protein between IIA and HPr with linker II, and pJFHI encoding a fusion protein between HPr and enzyme I with linker I (Schunk, 1992; Gerber, 1993). (^4)To facilitate purification, a hexahistidine tag was introduced at the carboxyl-terminal end of the fusion protein by polymerase chain reaction.


Figure 1: Plasmid map of pJFGCHI, linker sequence, and polyacrylamide gel of fusion protein. A, plasmid pJFGCHI encodes the fusion protein IICB-IIA-HPr-enzyme I under control of the Ptac promoter. Structure genes are indicated with solid boxes, and segments encoding the three Ala-Pro-rich linkers are indicated with open boxes. B, amino acid sequences of the three linker regions are indicated in boldface. Of the four subunits, only the amino-terminal and carboxyl-terminal sequences are shown. Residues in lower case indicate His Phe substitutions. Residues in square brackets are present in the wild-type protein but deleted in the fusion. C, lane1, molecular weight markers; lane2, membranes (100 µg); lane3, proteins removed by washing with Brij 35 (80 µg); lane4, Triton X-100-insoluble fraction (10 µg); lane5, Triton X-100-soluble fraction (5 µg); lane6, the fusion protein (2 µg) after Ni-nitrilotriacetic acid purification.



Purification of the IICB-IIA-HPr-Enzyme I Fusion Protein

E. coli ZSC112LDeltaHIC(pJFGCHI) was grown in 1 liter of LB at 37 °C to A = 1.5 and induced with 50 µM isopropyl-1-thio-beta-D-galactopyranoside, and incubation continued overnight. Cells were harvested by centrifugation (16,000 g at 4 °C for 10 min). The cell pellet (6 g) was resuspended in 18 ml of buffer A (10 mM Tris/HCl, pH 7.5, 500 mM NaCl, 1 mM beta-mercaptoethanol, 1 mM PMSF). Cells were broken by two passages through a French pressure cell. Cell debris was removed by low speed centrifugation (12,000 g at 4 °C for 10 min). Membranes were collected by high speed centrifugation (200,000 g at 4 °C for 2 h) and resuspended in buffer B (10 mM Tris/glycine, pH 9.3, 1 mM beta-mercaptoethanol, 1 mM PMSF; 1 ml/g of original cell paste). Brij 35 (Fluka, Buchs, Switzerland) was added to a final concentration of 2% to the stirred suspension, and, after 10 min of additional stirring, the membranes were collected by high speed centrifugation. The supernatant was discarded, and the membrane sediment was resuspended in buffer B (1 ml/g of original cell paste). These Brij 35-washed membranes were solubilized in 2% Triton X-100 (Serva, Heidelberg, Germany). After high speed centrifugation, the membrane sediment was extracted a second time under the same conditions. The pooled supernatants containing the solubilized fusion protein were mixed with 4 ml of Ni-nitrilotriacetic acid resin (equilibrated with buffer C; 10 mM Tris/HCl, pH 8.2, 500 mM NaCl, 1 mM beta-mercaptoethanol, 1 mM PMSF, 0.1% Triton X-100) and shaken for 1 h at 4 °C. The resin with the adsorbed protein was packed into a column, washed with 30 ml of buffer C and 30 ml buffer D (10 mM Tris/HCl, pH 7.5, 500 mM NaCl, 1 mM beta-mercaptoethanol, 1 mM PMSF, 0.1% Triton X-100, 20 mM imidazole) until A returned to base line. The fusion protein was then eluted with buffer E (10 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM beta-mercaptoethanol, 1 mM PMSF, 0.1% Triton X-100, 100 mM imidazole). The flow rate was set to 0.5 ml/min, and 2-ml fractions were collected. The fractions containing the fusion protein were dialyzed against buffer F (20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF, 0.1% Triton X-100) overnight at 4 °C.

Purification of Wild-type PTS Proteins

Enzyme I, HPr, and IIA were purified as described by Mao et al. (1995), IICB was purified as described by Waeber et al.(1993), and IIAB was purified as described by Stolz et al.(1993). IIC-IID was purified by metal chelate affinity chromatography. (^5)

Assay for Phosphoenolpyruvate:Sugar Phosphotransferase Activity

Phosphotransferase activity was assayed by the ion-exchange method of Kundig and Roseman(1971) as described by Erni et al.(1982). The concentrations of PTS proteins, of E. coliL-alpha-phosphatidylethanolamine (Type IX, Sigma), and of Triton X-100 are indicated in the figure legends.

Other Techniques

Protein samples were not boiled in sample buffer prior to electrophoresis. The IICB-IIA-HPr-enzyme I fusion protein was analyzed on a standard 10% polyacrylamide gel and stained with Coomassie Blue (Erni et al., 1982). For immunoblots (Towbin et al., 1979), monoclonal anti-IICB antibodies (Meins et al., 1988) and alkaline phosphatase coupled to anti-mouse IgG were used. Protein concentrations were determined by a modified Lowry assay (Markwell et al., 1978) with bovine serum albumin as standard.


RESULTS

Construction, Overexpression, and Purification of the IICB-IIA-HPr-Enzyme I Fusion Protein (GCHI Fusion)

The four genes ptsG crr ptsH, and ptsI were linked in the order by which their respective gene products mediate phosphoryltransfer. To maximize protein expression and minimize plasmid loss during fermentation, the gene was put under the control of the Ptac promoter (Fig. 1A). The four protein subunits were linked through Ala-Pro-rich linker peptides of 20 residues (Fig. 1B) taken from the IIAB subunit of the E. coli mannose transporter (Erni et al., 1987) and from the multiphosphoryl transfer protein subunit of the R. capsulatus fructose transporter (Wu et al., 1990). The GCHI fusion could be solubilized from Brij 35-washed membranes. As observed during purification of the mannose transporter, Brij 35 removed a large amount of other membrane (associated) proteins (Rhiel et al., 1994). 60% of the activity could be solubilized by extracting the membrane fraction twice with 2% Triton X-100. The fusion protein was purified to almost homogeneity by metal chelate affinity chromatography on a Ni-nitrilotriacetic acid column (Fig. 1C). Recoveries of 40% could be achieved using batch absorption instead of loading the extract to a prepacked column. 3 mg of pure GCHI protein were obtained from 6 g, wet weight, of cells. The fusion protein has a polyacrylamide gel electrophoretic mobility corresponding to a molecular mass of 140 kDa, which is in good agreement with the expected 150 kDa (Fig. 1C). The stability (enzymatic activity) of the fusion protein could be improved when imidazole used for elution was removed by dialysis. The column-bound fusion protein could be washed with and then eluted in detergent-free buffers without precipitation. The phosphotransferase activity in detergent-free buffer was only 2%. 70% of the full activity could, however, be recovered when detergent and lipids were added back to the incubation mixture. Attempts to concentrate the fusion protein in detergent-free buffer resulted in precipitation, indicating a limited solubility under these conditions.

Phosphotransferase Activity of the GCHI Fusion

E. coli ZSC112LDeltaHIC(pJFGCHI) fermented glucose on MacConkey plates, indicating functional complementation of the glucose phosphotransferase system by the fusion protein. The phosphoenolpyruvate-dependent glucose phosphorylation activity of the purified fusion protein is 3-4 times higher than that of an equimolar mixture of the isolated subunits (Fig. 2A). The nonlinear increase of activity with protein concentration is caused by Triton X-100, which is introduced with each aliquot of fusion protein (Fig. 2B). It does not reflect an exponential protein-activity relationship. The GCHI fusion protein and the free subunit system both require phospholipids for maximal activity, but the fusion protein is further stimulated by low concentrations of detergent. Fig. 3shows that a detergent lipid mixture is better than either pure phospholipid or pure detergent, while the free subunit system is inhibited by increasing concentrations of detergent (Fig. 2B). An optimal detergent-lipid ratio is required for maximum activity of the fusion protein. The GCHI fusion also catalyzes equilibrium exchange of phosphate between glucose and glucose 6-phosphate with 80% of the efficiency of the IICB subunit (Fig. 2C), indicating that IIA, HPr, and enzyme I in the fusion protein do not strongly interfere with substrate binding. In this case, however, the nonlinear protein-activity relationship is due to the inhibitory effect on transphosphorylation of increasing Triton X-100 concentrations (results not shown).


Figure 2: Phosphotransferase activity of the purified fusion protein and of an equimolar mixture of the isolated subunits. A, P-enolpyruvate-dependent phosphorylation of alphaMG by the fusion protein () and by the subunit mixture (). Triton X-100 concentration increased from 0 to 0.009% for the fusion protein, and from 0 to 0.003% for the subunit mixture. B, effects of Triton X-100 on P-enolpyruvate-dependent phosphotransferase activity of the fusion protein (40 nM, ) and of the subunit mixture (40 nM of each subunit, ). 100% activity corresponds to 3.90 nmol of alphaMG-P/30 min for the fusion protein and to 1.48 nmol of alphaMG-P/30 min for the subunit mixture. C, glucose 6-phosphate-dependent transphosphorylation of [^14C]Glc by the fusion protein () and by the purified IICB subunit (). Concentration of Triton X-100 increased from 0 to 0.027% for the fusion protein and from 0 to 0.009% for the IICB subunit. Shown are means and S.D. of three independent experiments. Assay conditions were as follows: E. coli lipids (Type IX, Sigma) 2 mg/ml; [^14C]alphaMG (DuPont NEN) 0.5 mM, 1000 dpm/nmol (A and B); [^14C]Glc (DuPont NEN) 1 mM, 550 dpm/nmol (C); glucose 6-phosphate 65 mM.




Figure 3: Dependence of phosphotransferase activity on Triton X-100/lipid ratio. The concentration of the fusion protein was 20 nM. Shown are means and S.D. of three independent experiments. Assay condition were as follows: [^14C]alphaMG 0.5 mM, 1000 dpm/nmol.



Channeling of Phosphoryl Transfer

The GCHI fusion protein has a 4-fold higher catalytic activity than a 1:1:1:1 mixture of the four subunits. This modest increase could be a consequence of two opposing effects: (i) high local concentration of active sites which accelerates phosphoryltransfer and (ii) steric constraints which cause non-optimal docking between phosphorylation sites and decrease of the phosphoryltransfer rate. To identify possible ``bottlenecks'' in the phosphoryltransfer chain, ``bypasses'' were opened by adding the subunits, one at the time, to the fusion protein and, for comparison, also to the 1:1:1:1 subunit mixture. Addition of IICB had a 2-3-fold stimulatory effect in both systems, suggesting that phosphoryltransfer between IIA and IICB and/or between IICB and glucose is rate-limiting (Fig. 4). This stimulatory effect is due to IICB itself. It cannot be accounted for by the increasing Triton X-100 concentration, which in this range would stimulate no more than a few percent (Fig. 2B). In contrast, addition of IIA and enzyme I in up to 7-fold molar excess to the fusion protein is slightly inhibitory (Fig. 4A). The reason for this effect is not understood. No inhibition occurs in the control assay with the single subunits (Fig. 4B).


Figure 4: Effects of extra IICB, IIA, HPr, and enzyme I on the phosphotransferase activity of the fusion protein (A) and of the subunit mixture (B). The concentrations of IICB (), IIA (), HPr (), and enzyme I () are indicated. A, 100% activity corresponds to 3.43 ± 0.54 nmol of alphaMG-P/30 min at 40 nM fusion protein. The Triton X-100 concentration increased from 0.010 to 0.012% when IICB was added. B, 100% activity corresponds to 1.03 ± 0.09 nmol of alphaMG-P/30 min at 40 nM subunit concentration. Triton X-100 concentration increased from 0.003 to 0.005%. Shown are means and S.D. of three independent experiments. Assay condition as in Fig. 2A.



Fusion proteins consisting of sequential enzymes can direct the substrate flow along the linked active sites and protect it from diversion into other metabolic pathways. In the phosphotransferase system, the phosphorylcarrier protein HPr acts as a branch point of phosphoryltransfer to the IIA domains of other PTS carbohydrate transporters. Is the HPr moiety in the GCHI fusion protein still accessible to free IIA units other than the covalently linked IIA moiety? The IIABbulletIICbullet IID complex and dGlc were added to the GCHI fusion and to the system of free subunits. As shown in Fig. 5, the mannose transporter complex did not interfere with the phosphotransferase activity of the GCHI fusion but competitively inhibited the activity of the free subunits. The 1.5-fold increase of the GCHI activity is due to the Triton X-100, which is introduced together with IICD and not to IICD itself. This suggests that channeling of the phosphoryl group transfer by the GCHI fusion occurs and that phosphorylgroups in the fusion are effectively protected from diversion into other phosphotransferase cascades.


Figure 5: Channeling of phosphoryl transfer in the fusion protein. Phosphorylation of [^14C]alphaMG by the fusion protein () and by an equimolar mixture of the isolated subunits () were shown. Activities of the fusion protein (40 nM) or the subunit mixture (40 nM of each subunit) were compared in the presence of an increasing concentration of the mannose transporter (IIAB, IICbulletIID) and 2.5 mM unlabeled 2-deoxyglucose. The concentration of Triton X-100 increased from 0.01 to 0.03% for the fusion protein and from 0.003 to 0.023% for the subunit mixture. 100% activity corresponds to 4.07 ± 1.10 nmol of alphaMG-P/30 min for the fusion protein and to 1.02 ± 0.04 nmol of alphaMG-P/30 min for the subunit mixture. Shown are means and S.D. of three independent experiments. Assay condition as in Fig. 2A.




DISCUSSION

Length and Stability of Linker Peptides

The four subunits of the glucose-specific phosphotransferase system were linked in the order by which they catalyze the phosphoryltransfer from phosphoenolpyruvate to glucose. The membrane-spanning IICB subunit was placed at the amino terminus in order to minimize possible interference with membrane insertion. The subunits were linked by Ala-Pro-rich linker peptides of 20 residues. If fully extended, this linker can span a distance of 6.5 nm, corresponding to the cross-section of spherical protein of M(r) 110,000. Ala-Pro linkers can function as universal joints between domains. They adopt a relatively stiffened conformation and cannot collapse to a random coil (Radford et al., 1989; Perham, 1991). While their amino acid sequence is unlikely to be important, length and composition may influence (i) proteolytic stability of the linker and (ii) activity of the linked subunits. Only a few very faint protein bands of lower electrophoretic mobility could be detected with a monoclonal anti-IICB antibody on Western blots of cell lysates (results not shown). With a polyclonal anti-IIA serum, a stronger smear of cross-reacting degradation products could be detected (results not shown) which, however, were removed by Ni chelate affinity chromatography. Doubling of the linker length (from 21 to 40 residues) between the IIA and HPr domain in the GCHI fusion resulted in markedly enhanced degradation by host proteases (results not shown). However, susceptibility to proteolysis is context-specific. A similar increase of the length of the linker was well tolerated in the two-domain IIAB subunit and the binary IICB-IIA fusion protein (Erni et al., 1989; Schunk, 1992). Taken together, the Ala-Pro-rich linker appears rather stable against endogenous proteases, while it turned out to be highly sensitive toward in vitro proteolytic cleavage (Erni et al., 1989). The length of the linker also affects phosphotransferase activity. Doubling its length increased the activity of the binary HPr-enzyme I fusion 1.4-fold; removal decreased activity 0.5-fold. Doubling the length did not affect the activity of the binary IICB-IIA fusion; removal decreased activity to 17% of the control with a 20-residue linker (Schunk, 1992; Schunk et al., 1992).

Activity of Fusion Protein and Possible Mechanism

The quaternary fusion protein is active in vivo and in vitro. The transport activity in vivo indicates that incorporation of the transmembrane IIC domain into the fusion protein did not compromise membrane insertion. The purified GCHI fusion has a 4-fold higher overall k than an equimolar mixture of the four subunits. This rules out the idea that the observed activity is only residual and due to proteolytic fragments generated from the fusion protein during purification. The quaternary fusion is more active than any of the binary fusions, suggesting a high degree of cooperativity between the domains and a strong compensation by proximity effects of potentially unfavorable steric constraints. For comparison, the binary HPr-enzyme I fusion has only 10% of the activity of an equimolar mixture of the separate subunits, the binary HPr-IIA fusion has less than 5%, and only the binary IICB-IIA fusion has a 4-fold higher activity than the 1:1 mixture (Schunk et al. 1992). The phosphotransferase activities of the HPr-enzyme I and IIA-HPr fusions could be stimulated by in vitro tryptic cleavage, suggesting unfavorable steric constraints in the fusion proteins (results not shown).

Because the IICB subunit occurs as a dimer (Erni et al., 1982; Meins et al., 1988) the fusion protein could dimerize, too. In this case, phosphoryltransfer could occur either along the domains within a monomer or between domains on different monomers. It is not known which mechanism is operative. Indicative of an intersubunit mechanism would be the negative dominance of a GCHI fusion with four inactive phosphorylation sites.

Fusion of the four subunits resulted in channeling of phosphoryltransfer such that the flow of phosphorylgroups could no longer be diverted from the HPr domain of the GCHI fusion protein toward the IIABbulletIICbulletIID complex. The addition of enzyme I, HPr, or IIA did not stimulate the overall phosphotransferase activity of the GCHI fusion protein, indicating that phosphoryl groups could not be funneled into the fusion protein, either. In contrast, the binary HPr-enzyme I fusion could be stimulated with extra HPr (Schunk et al., 1992), and the binary IICB-IIA fusion could be stimulated with extra IIA (Schunk, 1992). However, no stimulation of the binary IIA-HPr fusion by extra HPr or IIA occurred. (^6)

Unexpectedly, the phosphotransferase activity of the GCHI fusion was higher in a mixture of Triton X-100 and phospholipids than in either of the two components. Phospholipids are necessary for maximal activity of the transmembrane IICB subunit (Erni et al., 1982), while the detergent might prevent intramolecular aggregation of the crowded domains by loosening hydrophobic contacts. That the fusion protein tends to aggregate became manifest during attempts at keeping the GCHI fusion in detergent-free buffers. The protein could be eluted from the metal chelate column without detergent, but yields were less than 40% and subjected to high speed centrifugation (200,000 g at 4 °C for 20 min) it immediately sedimented. The protein also aggregated upon standing at 4 °C. Nevertheless, should the hydrophilic part (IIB-IIA-HPr-enzyme I), which accounts for 70% of the total mass, determine the behavior of the protein, working without detergents might be an option in attempts to crystallize the fusion protein.

Other Fusion Proteins of the Phosphotransferase System

Although no phosphotransferase system comprising all four phosphorylation sites and the transmembrane domain in a single polypeptide has been found in nature, fusion proteins comprising two or three modules exist. The most frequent natural fusions occur between IIA and IICB, e.g. in the mannitol transporter (IICBA) and the GlcNAc transporter (IICBA) (for reviews, see Erni(1992), Postma et al.(1993), and Lengeler et al.(1994)). In IICBA, channeling between IIA and IIB is not tight, and phosphorylgroups can also be transferred from the IIA domain of IICBA to the IIB domain of the glucose transporter (IICB) (Vogler et al., 1988). FPr of Salmonella typhimurium is a fusion protein comprising an HPr, a IIA, and a third domain exhibiting 60% similarity to the consensus receiver module of two-component regulatory systems (Geerse et al., 1989; Wu et al., 1990). Being part of the fructose transporter (IIBC) FPr nevertheless can complement HPr defects by transferring phosphorylgroups to IIA domains other than the one of FPr (Geerse et al., 1989). The most spectacular of naturally occurring fusion proteins is multiphosphoryl transfer protein of R. capsulatus, which comprises the enzyme I, HPr, and IIA domains belonging to the fructose transporter (Wu et al., 1990).

Conclusion

A number of recombinant bifunctional fusion proteins, which catalyze sequential reactions, have been reported. Compared with the separate enzymes in solution, they showed kinetic benefits for the overall reaction, e.g. shorter transient times and sequestering of the intermediate (Blow, 1987; Ljungcrantz et al., 1989; Lindbladh et al., 1994). The fusion protein reported in this paper, which consists of four independent but functionally related proteins, is apparently more complicated than any other that has been reported so far. It is not yet clear how the four components of the PTS interact with each other. Studies on interactions between the IIB and the IIA subunits by NMR are now in progress (Golic Grdadolnik et al., 1994). (^7)Our results are consistent with other observations on fusion proteins, suggesting that gene fusion technique may be a general and powerful method to construct fusion proteins that have novel properties. Crystallization of the fusion protein could be facilitated if IIA and HPr, known to crystallize, tend to dominate the behavior of the system and should the fusion protein behave more like a soluble than like a membrane protein. From a practical point of view, the use of artificial multifunctional enzymes (fusion proteins) will be useful in metabolic engineering. Multifunctional enzymes will help to direct substrates into a desired metabolic pathway in vivo. The GCHI fusion protein containing all components necessary for transport and phosphorylation is now being expressed in yeast to test whether it will function in an heterologous environment where no phosphotransferase system has been found.


FOOTNOTES

*
This work was supported by Grants 31-29795.90 from the Swiss National Science Foundation and the Deutsche Forschungsgemeinschaft (Er 147/1-2). 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: Inst. 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, phosphoenolpyruvate-sugar phosphotransferase system; IICB, transmembrane subunit of the glucose transporter; IIA, cytoplasmic subunit of the glucose transporter; HPr, histidine-containing phosphocarrier protein of the PTS; PMSF, phenylmethanesulfonyl fluoride; GCHI fusion, IICB-IIA-HPr-enzyme I fusion protein; IIC and IID, transmembrane subunits of the mannose transporter; IIAB, hydrophilic subunit of the mannose transporter; IICBA, the N-acetylglucosamine transporter; ptsG ptsH ptsI crr, genes coding for IICB, HPr, enzyme I, and IIA; alphaMG, methyl-alpha-D-glucopyranoside.

^2
M. Eberstadt and B. Erni, unpublished results.

^3
M. Manni and B. Erni, unpublished results.

^4
Q. Mao, unpublished results.

^5
M. Huber and B. Erni, unpublished results.

^6
Q. Mao, T. Schunk, B. Gerber, and B. Erni, unpublished results.

^7
M. Eberstadt, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. B. Stolz for advice and helpful discussions during this work.


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