THE JOURNAL OF B~LOGKXI, CHEMWIW 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 8, Issue of March 15, PP. 4254-4260, 1990 Printed in U. S A.

Overproduction, Solubilization, and Reconstitution of the Maltose Transport System from Esche&Ez coE*

(Received for publication, October 2, 1989)

Amy L. Davidson* and Hiroshi Nikaido From the Department of Mokcular and Cell Biology, University of California, Berkeley, California 94720

Maltose is transported across the cytoplasmic mem- brane of Escherichia coli by a binding protein-depend- ent transport system. We observed a lo-fold increase in the level of transport activity in assays with mem- brane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The ad- dition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hy- drolysis of ATP was directly coupled to maltose up- take. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining ex- periments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.

Maltose is transported into Escherichia coli by one of a class of binding protein-dependent active transport systems, each of which is composed of at least four distinct polypep- tides. In the case of the maltose transport system, five proteins are involved (Hofnung, 1974, Kellerman and Szmelcman, 1974, Silhavy et ul., 1979). The LamB protein forms a maltose- and maltodextrin-specific channel in the outer membrane (Szmelcman and Hofnung, 1975). The mulE gene encodes a periplasmic maltose binding protein (MBP)’ required for transport (Kellerman and Szmelcman, 1974). The MalF, MalG, and MalK proteins are believed to form a complex in the inner membrane. MalF and MalG are quite hydrophobic, and it is expected that they span the membrane (Dassa and Hofnung, 1985; Froshauer and Beckwith, 1984), while MalK

* This work was supported by United States Public Health Service Grant AI-09644. The costs of publication of this article were defrayed in part by the payment of pabe charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

i Sunnorted bv United States Public Health Service Postdoctoral Feilow&p GM-il717. To whom correspondence should be addressedz LSA Box 11, Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

’ The abbreviations used are: MBP, maltose binding protein; IPTG, isopropyl-p-D-thiogalactoside; LB, Luria broth; octyl glucoside, octyl- fl-D-glucopyranoside; SDS, sodium dodecyl sulfate.

is more hydrophilic and appears to be peripherally associated with the cytoplasmic surface of the membrane (Bavoil et al., 1980; Shuman and Silhavy, 1981). These genes are transcribed by two divergent operons (see Fig. 1) in the malB region at 91 min on the E. coli chromosome (Hofnung, 1974; Silhavy et ul., 1979).

Following a decade of uncertainty regarding the energetics of bacterial binding protein-dependent systems (Landick et ul., 1985), recent work has begun to clarify the question of the energy source for active transport. Both the E. coli maltose transport (Dean et ul., 1989b) and the SulmonelZu typhimu- rium histidine transport systems (Prossnitz et ul., 1989) have been reconstituted in right-side-out membrane vesicles in the presence of their respective binding proteins. In these systems transport is dependent on the presence of a proton-motive force which can generate ATP via the F,,-F1-ATPase. More significantly, maltose transport linked to ATP hydrolysis was detected in the absence of a proton-motive force when mem- brane vesicles from a strain carrying a deletion of the FOF1- ATPase (UC) were prepared in the presence of ATP (Dean et ul., 1989a).

A model in which the hydrolysis of ATP provides energy for maltose transport is consistent with the observation that the mulK gene contains a nucleotide binding domain (Higgins et ul., 1985). Two proteins from other binding protein-de- pendent systems which are homologous to MalK have been labeled with 8-azido ATP (Higgins et al., 1985; Hobson et ul., 1984), and binding of ATP to purified MalK has been dem- onstrated.* The homology between these proteins has recently been extended to include many other proteins putatively involved in transport processes (Albright et al., 1989) includ- ing the mammalian P-glycoprotein associated with multidrug resistance, which has been shown to hydrolyze ATP (Hamada and Tsuruo, 1988). It now appears likely that ATP hydrolysis by a component of the transport complex, presumably MalK, provides the energy required for active maltose uptake; how- ever, positive proof cannot be obtained using the vesicle system because crude membrane vesicles are capable of con- siderable metabolism (Dean et ul., 1989a; Hunt and Hong, 1983; Prossnitz et ul., 1989). The possibility that a metabolite of ATP rather than ATP itself interacts with the transport machinery cannot be discounted until transport activity and ATP hydrolysis can be detected in a purified reconstituted system.

In this study, we have made a major advance toward this goal by overproducing the inner membrane proteins, MalF, MalG, and MalK, in a functional form. We have succeeded in solubilizing the overexpressed proteins from the E. coli mem- brane and in reconstituting active maltose transport, depend- ent only on the presence of ATP, in phospholipid vesicles using a detergent dilution procedure (Ambudkar and Maloney,

’ H. Nikaido, unpublished data.

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Reconstitution of the Maltose Transport System

1986a). We expect that this system will serve as a useful model in which to study the structure and function of the maltose transport complex and as an assay for the purification of a functional complex.

4255

Labeling of MalF and MalG with [“S1Methionine-Strain K38 (HfrC (A)) (Russel and Model, 1984) containing pFGl9 was grown in minimal medium M63 (Miller, 1972) supplemented with 0.2% maltose and 5 pg/ml thiamine at 37 ‘C. At an AeW of 0.3, an Ml3 phage (mGPl-2, a gift of S. Tabor) carrying the T7 RNA polymerase gene under control of the lacUV5 promoter was added at a multiplicity of infection of 20, and expression of the polymerase was induced by adding 0.1 mM IPTG. After 30 min at 37 ‘C, rifampicin was added to a final concentration of 200 &ml. Thirty min later, cells were labeled with 10 pCi/ml [35S]methionine for 5 mm, then collected by centrif- ugation, and suspended in SDS-loading buffer for electrophoresis.

EXPERIMENTAL PROCEDURES &rains and &or&h Conditions-All strains used in this study were

derivatives of E. coli Kl2. An F’ factor carrying the 1acP mutation and Tn5 (kanamycin) was transferred to pop3327 (F- malF araD lot rp.sLl) (Debarbouille et al., 1978) using a standard mating tech- nique (Miller, 1972) to generate the strain HN596 (malF araD lac rpsLl/F’ lacZq locZ:Tn5, proA+proB+). Strain HN597 (malp AuncB- C ilv::TnlO araD lac rpsLl/F’ 1acP lncZ::Tn5, proA+ proB+) was derived from HN596 by introducing a deletion of the FOFi-ATPase (uric) via Pl cotransduction with the closelv linked ilu::TnlO from donor strain DK8 (Hfr PO1 AuncB-C il&TnlO bglR thi-Z rel-1) (Dean et al., 1989a). Strain HN594 (F- malF malBAl3 AuncB-C ilv::TnlO argH his rpsL1) was made by transduction of the same uric deletion into HNll5 (F- malF malBAZ3 argH his rpsLZ), which contained a deletion of most of the malB region including malK, malE, malF, and malG (Hofnung, 1974). In order to construct a strain carrying both the mazy and maZE24-1 mutations, a Tn10 insertion closely linked to rnal7’ was moved by Pl transduction from donor strain HN606 (F- malQfAm) zhg::Tn10 thi xyl mtl gaZ lacy ton4 kc) (Dean et al., 1989a) into strain ~0~3327. Since the rnaly allele causes constitutive expression of maltose-inducible operons, tetracycline- resistant transductants were screened for high rates of maltose trans- port in whole cells under noninducing conditions. This strain was saved as strain HN700. Because of the close proximity of mal2’ and malQ, this strain was also malQ+. The malp allele was then trans- ferred by Pl cotransduction with zhg::TnlO into strain CC142 (malE24-1 AlncUl69 araD139 rpsLl56 thi flbB5301 deo7ptsF25 relA/ F’ lacP Tn5 (Dean et al., 1989b) to generate strain HN701.

Strains were grown with aeration at 37 ‘C in LB (10 g of tryptone, 10 g of yeast extract, 5 g of NaCl/liter (Miller, 1972)) or 2 X LB (20 g of tryptone, 20 g of yeast extract, 5 g of NaCl/liter) as indicated. Antibiotics necessary to maintain plasmids were added at the concen- trations recommended by Maniatis (Maniatis et al., 1982). When cells reached a density equivalent to 0.2 Aco0 units, expression of the plasmid-encoded ma1 genes was induced by adding isopropyl B-D- thiogalactoside (IPTG) to a final concentration of 0.1 mM. Two hours later, cells were harvested for preparation of membrane vesicles.

Con&r&ion of P&smids-The expression vector pKK233-2, which contains a trp/lac fusion promoter (trc) and the 1acZ ribosome binding site upstream from unique NcoI and Hind111 restriction sites (Amann and Brosius, 1985), was used to increase expression of the malF and malG genes. DNA coding for malF and malG was obtained from the plasmid pMB3, a gift of M. Hofnung (Dassa and Hofnung, 1985). All manipulations of DNA were made using standard technfques (Man- iatis et al., 1982). The nlasmid DMB~ was cleaved with XhoII. and the 4.4-kilobase fragment carrying malF and malG (Fig. 1) was purified and ligated to pBR322 DNA digested with BamHI to generate a Hind111 site downstream of r&G. The resulting nlasmid was cut at a unique NcoI at the start site of the malF geneand at the Hind111 site to generate a 4.4-kilobase fragment which was isolated and ligated to DNA from pKK233-2 restricted with NcoI and HindIII. This plasmid, in which expression of both malF and malG were under control of the trc promoter, was called pFG23 (ampicillinr). A second plasmid, pFGl9 (ampicillin), was constructed using the vector pT7- 5, a gift of S. Tabor, which carries the T7 phage promoter upstream from a multiple cloning site. The plasmid pFGl9 consists of the 3.5- kilobase MluI-PuaII fragment car-rying ma?F and malG inserted into the multiple cloning site of pT7-5. The plasmid pMRl1 (chloram- phenicol’), which expresses the malK gene under control of the trc promoter, was a gift of H. Shuman (Reyes and Shuman, 1988).

FIG. 1. Structure of the malB region. Arrows indicate genes required for maltose transport, which are transcribed from divergent operons in the maZB region of the E. coli chromosome at 91 min. Only those restriction sites used in the plasmid constructions described in this paper are shown.

Electrophoresti-SDS-polyacrylamide gel electrophoresis was per- formed according to the method of Lugtenberg (Lugtenberg et al., 1975), and gels were stained with Coomassie Blue. For autoradiogra- phy, gels were dried under vacuum and exposed to Kodak X-Omat AR film.

ZmmunobZot A&y&-Following electrophoresis, proteins were transferred to nitrocellulose as described by Towbin (Towbin et al., 1979), For optimum transfer, 20% methanol was included in the transfer buffer when MalG was to be detected but was omitted when MalF or MalK were to be detected. Immunodetection of proteins was accomplished by using polyclonal anti-MalF, anti-MalG, or anti- MalK antiserum and alkaline phosphatase-conjugated anti-rabbit antibody (Sigma) as described (Blake et al., 1984). Antiserum was raised against overproduced proteins which had been partially puri- tied and electroeluted from SDS-polyacrylamide gels.’

Preparation of Membrane Vesicle-Right-side-out membrane ves- icles were prepared by osmotic lysis of spheroplasts as described previously (Dean et al., 1989a, Dean et al., 1989b). Vesicles were resuspended in 20 mM KP!, pH 6.2, 3 mM MgClz and assayed imme- diately for maltose uptake or frozen at -70 “C.

Solubilization and Reconstitution-The octyl glucoside (octyl-O-D- glucopyranoside) dilution procedure (Racker et al., 1979) as modified by Ambudkar and Maloney (1986a, 1986b) was used to reconstitute maltose transport in proteoliposome vesicles. To solubilize membrane proteins, thawed membrane vesicles (1 mg/ml) were incubated at 0 ‘C for 30 min with 20 mM KPi, pH 6.2, 5 mM MgClz, 1 mM dithiothreitol, 20% glycerol, and 1.1% octyl glucoside (Calbiochem). The mixture was subjected to centrifugation at 100,000 x g for 30 min at 4 ‘C, and the supernatant, containing approximately 50% of the initial protein, was stored on ice until use.

To prepare liposomes, crude E. coli phospholipids (Avanti Polar Lipids, Inc.) were washed with acetone and ether as described (New- man and Wilson, 1980), resuspended in 20 mM KPi, pH 6.2, and 2 mM 2-mercaptoethanol at 50 mg/ml, and stored at -70 C under nitrogen until use. Aliquots were thawed, mixed with O-100 mM NaATP, pH 6.2, as required and sonicated to clarity in a bath-type sonicator.

Typically, 90 ~1 of sonicated lipid was mixed with 450 ~1 of the supernatant fraction and the concentration of octyl glucoside adjusted to 1.1%. After 15 min on ice this mixture was diluted into 14 ml of 20 mM KPi, pH 6.2, 1 mM dithiothreitol, and O-20 mM ATP at room temperature. Proteoliposomes were isolated by centrifugation at 100,000 X g for 1 h at 4 “C, resuspended in 5 ml of 20 mM KP,, pH 6.2, and 3 mM MgClz and subjected to a second centrifugation step at 100,000 x g to decrease the external ATP concentration.

Transport Assays-To measure maltose accumulation, membrane vesicles or proteoliposomes were diluted in 20 mM KP1, pH 6.2, 3 mM MgCl* at room temperature to a final concentration of 0.2-l mg of protein/ml (vesicles) or 0.1-0.3 mg/ml (proteoliposomes). MBP was added to a final concentration of 10 pM except in assays using vesicles derived from the malE24-1 strain. The wild-type MBP used in these studies was purified by affinity chromatography (Ferenci and Klotz, 1978) and was dialyzed extensively to remove most of the bound maltose (Silhavy et al., 1975). Membrane vesicle assays were per- formed either in the presence or absence of 10 mM ascorbate and 0.1 mM phenazine methosulfate as indicated. The transport reaction was initiated bv adding 114Clmaltose (Amersham Corn.. snecific activitv. 130 PCi &mol) to~a~final concentration of 10 pMi and 25-~1 samples were removed at the specified times, filtered through a Millipore tilter (0.45-pm HA for membrane vesicles and 0.22-pm GSTF for proteo- liposomes), and washed with 5 ml of 50 mM LiClz. The tilters were dried and counted in a liquid scintillation counter using Aquasol as scintillant (Du Pant-New England Nuclear). All values were corrected for background counts/min on filters (lo-30% of total counts/min) in the presence of MBP and maltose only.

’ A. Davidson and E. Rosenberg, unpublished data.

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4256 Reconstitution of the Maltose Transport System

Assuy oj A ?‘P Concentratmn-Aliquots from parallel assays using nonradioactive maltose were removed to ice-cold 7.5% perchloric acid, and ATP concentrations were determined using an ATP biolumi- nescence HS kit (Boehringer Mannheim) as described previously (Dean et al., 1989a).

Protein Assoys-Protein concentrations were determined using BCA reagent (Pierce Chemical Co.) with bovine serum albumin as standard.

RESULTS

overproduction and Identification of Gene Products-In or- der to overproduce the proteins involved in maltose transport, the malF, malG, and malK genes were placed under control of the trc promoter (Amann and Brosius, 198.5) on multicopy plasmids. Plasmid pMRl1, which contains malK under con- trol of the trc promoter, was obtained from H. Shuman (Reyes and Shuman, 1988). It has been shown previously that MalK migrates at a molecular weight of 40,000-43,000 in SDS- polyacrylamide gels (Bavoil et al., 1980; Reyes and Shuman, 198& Shuman and Silhavy, 1981). When a strain carrying pMRl1 was grown and induced with IPTG, increased amounts of MalK were seen in the membrane fraction at i’kfr 43,000 (Fig. 2, lanes 2 and 3, upper arrow; see also Reyes and Shuman, 1988). The plasmid pFG23 which contains the malF and malG genes under control of the trc promoter was con- structed in order to overproduce the other two membrane- associated components of the transport system (see “Experi- mental Procedures”). Following induction of strain JMlOl (Alac pro supI thi/F’ traD36 proAB lacIq ZAMl5) (Messing, 1979) harboring pFG‘23, two proteins in the membrane frac- tion increased in intensity, one migrating at a molecular weight of 43,000 and the other at 27,000 (Fig. 2, lunes 4 and 5, arrows). The true molecular weights of MalF and MalG as determined from the nucleotide sequences are 57,000 and 33,000, respectively (Dassa and Hofnung, 1985; Froshauer and Beckwith, 1984). To verify that the observed proteins are the products of the malF and malG genes, plasmid pFGl9 was constructed which contained the two genes downstream of

123456

FIG. 2. Overproduction and identification of MalF, MalG, and MalK. Strain HN597 (pMRl1) was grown in LB with chlor- amphenicol and kanamycin. Strain JMlOl was grown in minimal medium M63 containing 0.2% maltose, 0.2% glycerol, 5 fig/ml thia- mine, 0.1% casamino acids, and ampicillin. IPTG was added to a part of the cultures as described under “Experimental Procedures.” Cells were harvested by centrifugation, washed in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and lysed by sonication for 2 min with a Gallenkamp Soniprep 150 probe sonicator. Following a low speed (5,000 X g) centrifugation, the membrane fractions of the cells were collected by centrifugation at 100,000 x g for 1 h, and constant amounts (20 pg) of membrane protein were applied to a SDS-polyacrylamide gel. ,Qzne 1, molecular weight markers as indicated (X lo-‘); lun.e 2, HN597 (pMRl1) without IPTG; lane 3, NH597 (pMRl1) with IPTG; lone 4, JMlOl (pFG23) without IPTG; lane 4, JMlOl (pFG23) with IPTG. Lune 6 is an autoradiograph of total cellular protein from K38 (pFGl9) labeled with [35S]methionine as described under “Experi- mental Procedures.” Arrows indicate positions of migration of MalF and MalK (upper arrow) and MalG (lower urrow).

the T7 promoter (see “Experimental Procedures”). The spec- ificity of the T7 RNA polymerase for the T7 promoter coupled with the ability to inhibit selectively the E. coli RNA polym- erase with rifampicin permits the exclusive expression and labeling of genes under control of the T7 promoter (Tabor and Richardson, 1985). The autoradiograph in Fig. 2 (lane 6) shows that two proteins which corn&rate with the putative gene products in lane 5 were labeled with [35S]methionine. To confirm that the larger band was MalF, we deleted the malF gene extending from the ribosome binding site to the Sac1 site in pFGl9 (Fig. 1). In labeling experiments with this deletion plasmid, only the Mr 27,000 band was observed (not shown). These results are in agreement with a previous report in which the MalF protein was identified immunochemically by a gene fusion technique as a protein migrating at M* 40,000 in SDS-polyacrylamide gels (Shuman et al., 1980).

Maltose Transport in Vesicles-The current model for malt- ose transport is that the three proteins, MalF, MalG, and MalK, form a complex in the membrane which mediates maltose uptake into the cytoplasm. We were interested to see if simultaneous overproduction of all three proteins would increase the rate of maltose transport. In addition to its role in transport, MalK has a function in regulation of ma1 gene expression; overexpression of MalK prevents expression of the ma1 regulon (Reyes and Shuman, 1988). Therefore, these experiments were performed in strains carrying the malT mutation in which ma1 expression is constitutive and inde- pendent of MalK regulation (Reyes and Shuman, 1988).

Cells were transformed with plasmids pMRl1 and pFG23 simultaneously and were grown in the presence of ampicillin, chloramphenicol, and kanamycin to maintain both plasmids and the F’ factor. Membrane vesicles competent for maltose transport were prepared (Dean et al., 1989b), and maltose accumulation was measured. Table I presents the initial rates of maltose uptake into membrane vesicles prepared from strains grown with or without the plasmids and in the pres- ence or absence of IPTG. In strain HN596 which produces wild type MBP, the maltose transport activity resulting from constitutively expressed chromosomal ma1 genes was 0.2 nmol/min/mg protein. The addition of plasmids pMRl1 and pFG23 increased the activity 2-fold in the absence of the inducer IPTG. Following induction with IPTG there was a

TABLE I Initial rute of [W]maltose uptake into membrane vesicles

Strains were grown in LB (HN596, HN597, HN701, HN701 plus plasmids) or 2 x LB (HN596 plus plasmids, HN597 plus plasmids) and induced as described under “Experimental Procedures.” Mem- brane vesicles were prepared by shocking into 0.5 mM (HN596 and HN701) or 8 mM (HN597) ATP. Assays were performed by incubating vesicles with MBP (HN596 and HN597 only), ascorbate/phenazine methosulfate (HN596 and HN701 only), and [*4C]maltose. Aliquots were filtered at 10-s intervals to determine transport activity.

Strain Relevant genotype

Plasmids Transport pFG23, IPTG pMRl1 activity

HN596 ZUX+ malE+ HN596 HN596

nmol/min/mg protein

- - 0.228 + - 0.434 + i- 1.83

HN701 uric+ mulE24-1 - 5.04 HN701 + - 17.6 HN701 + + 30.4

HN597 uric- mulE+ - - 0.250 HN597 + - 0.295 HN597 + + 0.870

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Reconstitution of the Maltose Transport System 4257

further 4-fold stimulation of activity. It should be noted that when MalF, MalG, and MalK are overexpressed in a mulT+ strain, transport in membrane vesicles was the same as that seen in the malT strain presented here (data not shown). This is in contrast to what was seen in whole cells; when the proteins were overproduced, maltose transport was seen only in the presence of the malT mutation.

Serial dilutions of membrane vesicle preparations were analyzed by immunoblotting to obtain a crude estimate of the degree of overproduction of the ma1 proteins. In the absence of IPTG, the addition of plasmids pFG23 and pMRl1 to strain HN596 resulted in a 2-fold increase in MalK protein associated with membrane vesicles. Following induction with IPTG, membrane vesicles from HN.596 (pMRl1, pFG23) con- tained 8 times more MalK than membranes from HN596. The degree of overproduction of MalF and MalG was slightly greater. Vesicles from strain HN596 (pMRl1, pFG23, no IPTG) had four times more MalF and MalG protein than vesicles from strain HN596; following induction, there was 16 times more MalF and MalG than in vesicles from HN596. These estimates agree well with the increase in activity in membrane vesicles. Since the transport activity of intact cells is recovered without much inactivation in membrane vesicles (Dean et ul., 1989b), the close correlation between increased activity and increased protein content seen here suggests that most of the overproduced protein which is recovered in mem- brane vesicles is in a functional form.

Maltose transport was also measured in a strain carrying the malE24-1 mutation (HN701). Rather than being secreted into the periplasm, the MBP in strain HN701 is tethered to the membrane via an uncleaved signal sequence, and it re- mains functional for transport assays in membrane vesicles (Dean et al., 1989b). The -fold increase of maltose transport in HN701 as a result of overproduction is similar to that seen in strain HN596, but the activities are substantially higher (Table I). The transport activities in strain HN597 which carries the uric deletion and wild type MBP were of the same magnitude as the activities in the U~C+ strain HN596 (Table 11.

Solubilization and Reconstitution of Overexpressed Pro- teins-Recent work on the reconstitution of membrane pro- teins suggests that the presence of excess phospholipid (New- man and Wilson, 1980) and glycerol (Ambudkar and Maloney, 1986b) during solubilization with octyl glucoside protects against denaturation, and this procedure has been used suc- cessfully to reconstitute many different transport systems (Ambudkar et al., 1986; Ambudkar and Maloney, 1986b; D’Souza et ul., 1987; Newman and Wilson, 1980; Tsuchiya et al., 1982). We therefore selected this technique for use in our studies with the E. coli maltose transport system. Briefly, membrane vesicles were incubated with octyl glucoside in the presence of glycerol to solubilize membrane proteins (exoge- nous phospholipid in this step was not required with the ma1 proteins). Following a centrifugation step, the octyl glucoside supernatant was mixed with bath-sonicated E. coli phospho- lipid in a small volume and then diluted 25-fold to decrease the concentration of detergent sufficiently that the hydropho- bic proteins would be incorporated into phospholipid vesicles. For the reconstitution experiments, we used strain HN597 (pMRl1, pFG23), which carries a deletion in most of the uric operon. The uric strain was selected so that ATP trapped inside proteoliposomes would not be hydrolyzed. Fig. 3 shows the protein composition of membrane vesicles of strain HN597 (pMRl1, pFG23) from both uninduced (he 2) and induced cells (&e 3). The MalF and MalK proteins corn&ate on the gel and appear as a prominent band at Mr 43,000 (lane

123 4

a MalG CI MalK a -t&IF nnn

5 6 7 8 9101112 -- -.j

22- w3lA

14-

FIG. 3. Solubilization of overproduced proteins and recon- stitution in liposomes. Lune 1 contains molecular weight markers (X lo-‘). NH597 (pMRl1, pFG23) was grown in 2 x LB with appropriate antibiotics, IPTG was added to a part of the culture, and membrane vesicles and proteoliposomes were prepared as described under “Experimental Procedures.” Constant amounts (20 pg) of pro- tein were loaded in lanes 2-6. Lane 2, SDS-polyacrylamide gel elec- trophoresis of memhrane vesicles prepared from sample without IPTG; lone 3, membrane vesicles prepared from sample with IPTG; lone 4, supernatant fraction following treatment of vesicles seen in lune 3 with octyl glucoside and centrifugation at 100,000 X g; lane 5, pellet fraction following treatment of vesicles with octyl glucoside and centrifugation at 100,000 X g; lane 6, reconstituted proteolipo- some vesicles. Four-pg samples of the preparations in lanes 2 and 3 were transferred to a nitrocellulose membrane and prohed with a I:500 dil.ution of antibody to MalF, MalG, or MalK as indicated. Lanes 7, 9,, and 11, HN597 (pMRl1, pFG23) without IPTG; lanes 8, 10, and 12, HN597 (pMRl1, pFG23) with IPTG. ATTOWS indicate positions of migration of MalF and MalK (upper CWOUJ) and of MalG (lower arrow).

3, upper urrow). Immunoblot analysis of these preparations was performed to demonstrate that all three proteins are overproduced in membrane vesicles following induction with IPTG (Fig. 3, lunes 7-12). Proteoliposomes were prepared following the procedure detailed under “Experimental Proce- dures;” lanes 4-6 demonstrate that the ma1 proteins are solu- ble in octyl glucoside in the presence of M$+ and glycerol and that the proteins are incorporated into the proteoliposome vesicles. An enrichment of the transport proteins during the preparation of proteoliposomes is apparent in this figure (compare lunes 3 and 6).

Maltose Transport in Proteoliposome Vesicles-We have been able to demonstrate maltose transport in proteolipo- somes reconstituted from E. coli phospholipids and solubilized membrane protein. Fig. 4 shows that maltose accumulation was dependent on the presence of MBP and ATP and was mediated by the products of the malB region. No transport was observed when proteoliposomes were prepared in the absence of ATP or when prepared from strain HN594, which carries a deletion of the malF, malG, and malK genes (Fig. 4). The activity of maltose uptake observed here, 3 nmol/min/ mg protein, is similar to the activity in membrane vesicles assayed with wild-type MBP. The slightly higher rate can be attributed to purification of the MalF-MalG-MalK complex during solubilization and reconstitution. Assuming an inter- nal volume of 15 pl/mg protein (calculated from data in Ambudkar and Maloney, 1986b), maltose was accumulated to a concentration of approximately 0.3 mM during the period of assay which reflects significant concentration of maltose over than in the assay medium (10 PM).

ATP Hydrolysis and Maltose Transport in Proteolipo- somes-The most convincing evidence to date that ATP provides the energy for maltose uptake comes from measure- ments of ATP in membrane vesicles derived from uric strains (Dean et al., 1989a). In vesicles prepared in the presence of

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TIME (min)

FIG. 4. Maltose accumulaCon in proteoliposomes. Membrane vesicles prepared from HN597 (pMRl1, pFG2.3) with IPTG or HN594 were thawed on ice, and proteoliposomes were prepared as described under “Experimental Procedures.” When ATP was included, phos- pholipid was sonicated in the presence of 20 mM ATP, and the dilution medium contained 20 mM ATP. Accumulation of [%]malL ose was measured in proteoliposomes as described. Symbols: 0, HN597 (pMRl1, pFG23) plus ATP; 0, HN597 (pMRl1, pFG23) no ATP; n , HN597 (pMRl1, pFG23) plus ATP, no MBP; A, HN594 plus ATP.

ATP, a stimulation of ATP hydrolysis was seen upon the addition of maltose. The rate of ATP hydrolysis varied and was from 4-10 times greater than the rate of maltose accu- mulation (Dean et ul., 1989a). We have now performed similar experiments in proteoliposomes prepared from strain HN597 (pMRl1, pFG23). In all experiments performed, both ATP hydrolysis and maltose uptake in proteoliposomes were stim- ulated by the addition of MBP to the assay medium, and ATP hydrolysis ceased when the vesicles stopped accumulating maltose (Fig. 5, B and C). As expected, in the absence of MBP, no ATP hydrolysis or maltose uptake was seen. Fig. 5A shows that no ATP hydrolysis or maltose transport was seen either in the presence or absence of MBP in proteoliposomes prepared from the mulB deletion strain HN594. In the exper- iment presented in Fig. 5B using strain HN597 (pMRl1, pFGZ3), 1.4 mol of ATP was hydrolyzed per mol of maltose accumulated. The low transport activity seen in this experi- ment as compared to the experiment in Fig. 4 is a function of the low and presumably subsaturating ATP concentration used here. The experiment presented in Fig. 5C is represent- ative of another set of experiments in which approximately 10 mol of ATP was hydrolyzed for every mol of maltose accumulated in the proteoliposomes. The 1O:l stoichiometry is typical of experiments performed in membrane vesicles in this laboratory (Dean et al., 1989a). When the initial ATP content of vesicles was high (>20 nmol/mg protein), the stoichiometry was usually high, with an average of 17 ATP hydrolyzed per maltose transported in five experiments. When the initial ATP concentration was low (~15 nmol/mg protein), in 8 out of 10 experiments the ratio was within the range of 0.8-1.8, with a mean of 1.4 mol of ATP hydrolyzed per mol of maltose transported. However, this correlation did not always hold, and both high and low stoichiometries were seen in vesicles containing intermediate concentrations of ATP. In both types of experiments, no accumulated [14C] maltose leaked from the vesicles over a 2-min period when a lOOO-fold excess of cold maltose was added to the assay mixture (not shown); hence the differences are not related to

ok-* - * - I 0 1 2

TIME (min)

FIG. 5. ATP hydrolysis and maltose accumulaGon in proteo- liposomes. Membrane vesicles prepared from HN597 (pMRl1, pFG23) with IPTG or HN594 were thawed on ice and proteoliposomes were prepared as described under “Experimental Procedures.” Phos- pholipids were sonicated in the presence of 40 mM ATP and diluted into medium without ATP. [%]Maltose uptake into proteoliposomes and ATP hydrolysis were measured in parallel assays as described under “Experimental Procedures.” Values are means k S.D. for n = 3 determinations. A, transport and hydrolysis in proteoliposomes prepared from HN594. B, transport and hydrolysis in proteoliposomes prepared from HN597 (pMRl1, pFG23); example of low rate of ATP hydrolysis. C, transport and hydrolysis in proteoliposomes prepared from HN597 (pMRl1, pFG23); example of high rate of ATP hydrol- ysis. Symbols: 0, ATP hydrolysis in the presence of MBP; 0, ATP hydrolysis in the absence of MBP; n , [%]maltose uptake in the presence of MBR A, [14C]maltose uptake in the absence of MBP.

leakage of maltose from the vesicles. The significance of these results will be discussed below (see “Discussion”).

DISCUSSION

In this report, we have overproduced the MalF, MalG, and MalK proteins in a functional form and have been able to solubilize the proteins from the membrane and reconstitute maltose transport activity in proteoliposome vesicles. The successful reconstitution of maltose transport represents a major advance in the study of binding protein-dependent transport systems. In agreement with the results of Reyes and Shuman (Reyes and Shuman, 1988) overproduction of MalK prevented expression of maltose-inducible genes in the ab- sence of the n&Y mutation as judged by the complete absence of transport in whole cells. In a malT+ strain, expres- sion of malK, m&F, and malG under control of the trc pro- moter was sufficient to obtain maltose transport in membrane vesicles with added MBP. These results show that no other maltose-inducible gene product is required for maltose trans- port.

The high maltose transport activity seen in vesicles from the mulE24-1 strain HN701 is probably a more accurate reflection of the transport capacity of the membrane complex. In the situation where the MBP is tethered to the membrane vesicle, the local concentration of MBP at the membrane surface would greatly exceed that in assays with wild-type

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Reconstitution of the Maltose Transport System 4259

MBP. The concentration of MBP in the periplasm in fully induced cells is approximately 1 mM, and it has been shown that 90 pM MBP is required for one-half maximal transport activity in whole cell studies (Manson et al., 1985). It is therefore likely that interaction of MBP with the membrane is limiting in our assays of maltose transport using 10 PM concentrations of wild-type MBP.

In our experiments, we found that the MalF, MalG, and MalK proteins from strain HN597 (pMRl1, pFG23) were soluble in the nonionic detergent octyl glucoside. In contrast, Reyes and Shuman reported that MalK protein, when over- expressed by itself, was essentially insoluble in nonionic de- tergents (Reyes and Shuman, 1988) We have independently observed the same result with MalK4 and have noted that MalF and MalG, when overproduced in the absence of MalK, are also insoluble under the conditions reported in this paper. The fact that the simultaneous overproduction of all three proteins altered the solubility properties of these three pro- teins suggests that these proteins interact in the membrane. It may be that the proteins are extracted from the membrane by octyl glucoside as a complex, rather than as individual proteins. These experiments provide the first biochemical evidence that the membrane-bound proteins may interact in the membrane.

We have also shown that maltose uptake into proteolipo- somes is dependent on ATP, MBP, and the gene products of the malB region (i.e. malF, malG, and/or malK). The proteo- liposome provides a better model system than crude mem- brane vesicles in which to study transport processes because it is less contaminated by both cytoplasmic and membrane proteins. The fact that ATP alone is sufficient to energize maltose uptake in this cleaner system strengthens the hy- pothesis that ATP is hydrolyzed by some component of the transport complex, presumably MalK, during the transport process. We expect that it will be possible to demonstrate ATP hydrolysis by the purified reconstituted system in the near future.

Based on anaerobic growth yield experiments in intact cells, it has been reported that cells consume an amount of energy equivalent to the hydrolysis of one ATP for the transport of one molecule of maltose (Muir et al., 1985). In the proteoli- posome experiments, we have been able to detect ATP hy- drolysis which is dependent on maltose uptake. However, while we have detected stoichiometries close to 1 mol of ATP hydrolyzed per mol of maltose accumulated in 70% of the experiments (see Fig. 5B), we do not obtain this result in all experiments and therefore do not claim to have duplicated the in uiuo results. In some experiments (see Fig. 5C), the rate of ATP hydrolysis is considerably higher than the rate of maltose accumulation. The same high stoichiometry was consistently seen in our laboratory when using membrane vesicles (Dean et al., 1989a). We suspect that subtle and as yet undetected variations in our preparation of proteolipo- somes may alter the interactions between subunits in the complex so that, while hydrolysis remains coupled to trans- port, some futile cycling may occur. We believe that this effect is reversible since proteoliposomes with low stoichiometry are prepared from membrane vesicles which have a high stoichi- ometry. There is also a possibility that this variable stoichi- ometry is an intrinsic property of a transport system of this type. It is well known that maltose can be accumulated over a lOO,OOO-fold concentration gradient (Boos and Hengge, 1983). In contrast, proton-motive force-dependent transport systems can achieve only about a 2000-fold concentration, because they will reach a thermodynamic equilibrium at this

’ H. Nikaido, unpublished data.

point with the driving force of the proton-motive force. It is an attractive possibility that as the maltose concentration gradient increases, the system may be able to adjust the stoichiometry to increase the driving force against the maltose gradient so that a very high concentration ratio can be achieved.

Another possibility is that the variable stoichiometry may have a function in regulation. It is known that glucose is metabolized in preference to maltose; the presence of glucose inhibits transcription of the ma1 operons even in the presence of maltose (Schwartz, 1987). This effect is mediated in part by a mechanism known as catabolite repression which re- sponds to energy availability in the cell. It has also been proposed that the enzyme IIIg’“co~ acts directly on MalK to inhibit maltose transport (Schwartz, 1987). Varying the stoi- chiometry could be another mechanism by which the cell can decrease the rate of maltose transport and regulate the inter- nal concentration of inducer to augment the effect of catab- olite repression.

Ackrzoz&dgrnents-We would like to thank David Dean for his helpful discussions and his assistance in the preparation of this manuscript. We thank Dr. Howard Shuman for the gift of the plasmid pMRl1 and Dr. Maurice Hofnung for the gift of strains and of the plasmid pMB3. We also thank Dr. Stanley Tabor for the gift of plasmids, strains, and phage required for the use of the T7 expression system.

Note Added in Proof-Since the submission of this manuscript, a paper describing the successful reconstitution of another binding protein-dependent transport system in proteoliposomes has appeared Bishop er ul. (Bishop, L., Agbayani, R., Jr., Amhudkar, S. V., Malonev. P. C.,.and Ames, G:, F.-L.-(1989) Pk. gatl. Acad. &i. lJ.‘S. A. 86; 6953-6957) used an octyl glucoside dilution method to incorporate the membrane-associated proteins of the histidine transport system of Salmon&z typhimurium into liposomes and showed that ATP is consumed concomitant with transport of the substrate. These results are in complete agreement with much of our data, obtained by the reconstitution of another transport system.

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A L Davidson and H Nikaidofrom Escherichia coli.

Overproduction, solubilization, and reconstitution of the maltose transport system

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