Functional Expression of the HumanMDR1Gene inEscherichia coli

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Vol. 333, No. 1, September 1, pp. 66–74, 1996 Article No. 0365

Functional Expression of the Human MDR1 Gene in Escherichia coli Anthony M. George,1 Mary W. Davey, and Alain A. Mir Department of Cell and Molecular Biology, University of Technology Sydney, Sydney, New South Wales 2007, Australia

Received May 6, 1996

In this preliminary study, we report the cloning of the human MDR1 cDNA into a prokaryotic expression vector and the consequent functional expression of heterologous P-glycoprotein in Escherichia coli. We demonstrate increased resistance to the P-glycoprotein substrates TPA/, TPP/, and puromycin; reduced accumulation of TPP/ and tetracycline by resistant cells; and the expression of a full-length immunoreactive P-glycoprotein molecule in the membrane fraction of resistant cells. The obvious structural and functional similarities of P-gp to prokaryotic ABC transporters and other efflux transporters argues for a more complete study of the consequences pertaining to the expression of human P-glycoprotein in E. coli. q 1996 Academic Press, Inc.

Key Words: P-glycoprotein; multidrug resistance; transport; Escherichia coli.

The success of chemotherapy in the treatment of cancer is frequently hampered by the development of multidrug resistance which has become increasingly identified in a wide variety of newly diagnosed and recurrent human tumors (1). Both in vivo and in vitro studies have demonstrated that this resistance is primarily associated with the expression of the human MDR12 gene product, P-glycoprotein (2, 3). This 170-kDa membrane protein confers cross-resistance to many structurally and functionally unrelated natural product drugs including Vinca alkaloids, anthracyclines, epipodophyllo1

To whom correspondence should be addressed at Department of Cell and Molecular Biology, University of Technology Sydney, Box 123, Sydney, NSW 2007, Australia. Fax: (612) 9514 4003. E-mail: [email protected]. 2 Abbreviations used: P-gp, P-glycoprotein; MDR1, human multidrug resistance gene; mdr1, mouse multidrug resistance gene; TPP/, tetraphenylphosphonium ion; TPA/, tetraphenylarsonium ion; IC50 , concentration that inhibits the growth rate by 50%; CCCP, carbonyl cyanide–chlorophenyl–hydrazone).

toxins, actinomycin D, and peptide antibiotics (4, 5). The increased expression of P-gp in the plasma membrane and decreased intracellular drug accumulation are the most consistent changes detected in cells that exhibit MDR, and P-gp is thus thought to catalyze the ATP-dependent efflux of cytotoxic drugs (6). P-gps are encoded by a small family of closely related genes which has three members in rodents (mdr1, mdr2, mdr3) and two in humans (MDR1, MDR2) (7). P-gp is a member of the ABC superfamily of ATPdependent transport proteins, present in both prokaryotes and eukaryotes, that regulate the trafficking of diverse molecules and the extrusion of toxins across biological membranes (8–10). The similarity of the structure of P-gp to other ABC proteins would appear to support a role for P-gp as an efflux pump, but its exact physiological function and substrate specificity are still ill-defined (11). Unlike other members of the ABC transporter family and close MDR homologs, Pgp is unique in having a broad substrate range. The mechanism by which P-gp effluxes a diverse range of substrates is largely an unresolved and controversial debate (7, 11). The difficulties encountered in examining the postulated role of P-gp in the efflux of cytotoxic drugs may be overcome in part by introducing and expressing the MDR1 gene in a prokaryotic host such as Escherichia coli. Such an idea seems feasible in view of the common ancestry of mammalian MDR genes and bacterial genes encoding ABC transporters (9, 12, 13). Thus, E. coli could be a reasonable reaction vessel and model for the study of P-gp-mediated transport in cells and vesicles. The cloning and functional expression of the murine cDNA mdr1 gene in E. coli has been reported (14); and follow-up studies on the membrane topology of the P-gp protein in E. coli suggest a revision (15, 16) of the model predicted by hydropathy analysis (2, 4). In this study, we report the cloning of the human MDR1 cDNA into a prokaryotic expression vector and the consequent functional expression of P-gp in E. coli.

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This may provide opportunities for the further study of the structure and mechanism of action of P-gp using E. coli as a paradigm for P-gp-mediated transport. MATERIALS AND METHODS Materials. Tetraphenylphosphonium bromide (TPP/) and tetraphenylarsonium chloride (TPA/) were purchased from Aldrich. Carbonyl cyanide–chlorophenyl–hydrazone was purchased from ICN Biomedicals Inc. C219 monoclonal antibody was from Centocor Corp. (Philadelphia, PA). Tetra-[3H]phenylphosphonium bromide (23 Ci/ mmol) and L-[2,3-3H]proline (28 Ci/mmol) were purchased from Amersham, and [7-3H]tetracycline (0.6 Ci/mmol) was from DuPont NEN Research Products. All other chemicals were of analytical or molecular biology grade. Plasmids and bacterial strains. pMDR2000XS (17) was obtained courtesy of Michael Gottesman. It contains the full-length cDNA for the human MDR1 gene on a 4380 SacI–EcoRI fragment in the vector pGEM2. The prokaryotic expression vector pPOW B2 (18) has tandem l PRPL promoters just upstream of a ribosomal binding site and restriction sites, and a lcIts857 repressor gene that is divergently aligned with the promoters (Fig. 1). It was generously provided by Barbara Power (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia). The E. coli strains used were: JM109 (recA1, supE44, endA1, hsdR17, gyrA96, relA1, thi, D[lac-proAB], F*[traD36, proAB/, lacIq, lacZDM15]); and UT5600 (19) (F0, ara, leu, azi, lacY, proC, tsx, entA, D[ompT 0 fepC], glnV[AS], trpE, rfbD, rpsL, xylA, mtl, thi, l0), obtained from the E. coli Genetic Stock Center (Yale University, New Haven CT). DNA methods. DNA preparations, restriction digests, recovery of DNA fragments from agarose gels, ligations, and filling-in of recessed termini with Klenow polymerase were performed as previously described (20). Preparation of electrocompetent cells and electroporation of DNA were performed according to the manufacturer’s instructions, using an E. coli Pulser (Bio-Rad). Double-stranded plasmid DNA was sequenced using a Sequenase 2.0 kit (United States Biochemicals) according to the handbook. A 3.0-kb EcoRI fragment of the MDR1 cDNA from pMDR2000XS was used as the probe for Southern hybridization with plasmid DNA restriction fragments blotted to a nylon membrane (Hybond-N/, Amersham). The probe was labeled with horseradish peroxidase using an Amersham ECL gene labeling and detection kit. Growth and induction of cells. Overnight cultures of E. coli containing either pAMG5 or pAMG6 were diluted 1:100 in LB broth supplemented with ampicillin (20 mg/ml), and grown with aeration at 220 rpm at 307C to mid-log phase and then subdivided and induced at various temperatures between 36 and 427C. For the drug challenge tests, cells were grown at 307C to an absorbance of 0.2 at 600 nm, induced at 407C for 30 min and then subdivided and their growth at 307C was monitored by absorbance at 600 nm, following the addition of given concentrations of drugs. The drug IC50 is the concentration that inhibits the growth rate by 50%. Western blots. Induced or uninduced cells were harvested, washed once in 10 mM KPi buffer (pH 7.5) containing 2 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride, and then resuspended in the same buffer containing 10% glycerol. These suspensions were disrupted by sonication, centrifuged at 47C for 10 min at 2500g to remove intact cells and then at 47C for 15 min at 12,000g to pellet the membrane fraction. The resuspended membranes were incubated with DNase I and RNase A for 15 min at 377C, then pelleted as previously, and frozen in dry ice/ethanol and stored at 0807C until required. Thawed membrane pellets were mixed with sample buffer at a protein concentration of 1 mg/ml (21), left at room temperature for 4 min, prior to electrophoresis in 5.6% polyacrylamide containing 9 M urea and 1.0% SDS (22). Immunoblotting and detection with the C219 monoclonal antibody were as described previously (23,

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24). The apparent molecular size of P-gp was calculated from its mobility relative to high-molecular-weight standards (Bio-Rad Laboratories). Transport assays. Overnight LB cultures of UT5600 and UT5600(pAMG6) were diluted 1:100 into fresh medium and grown with aeration at 307C to an A530nm of approximately 0.4 to 0.5. Half of each culture was left at 307C and the other half was induced at 427C for 15 min. The cultures were chilled and harvested, washed with 1 vol of ice-cold 50 mM KPi buffer (pH 7.5) and then washed again in the same buffer containing 10 mM EDTA (14). Cell pellets were then resuspended to an A530nm of 10 (equivalent to about 2 mg protein/ml) in 50 mM KPi buffer (pH 7.5) containing 10 mM MgSO4 . The uptake of [3H]TPP/ (23 Ci/mmol) at a final concentration of 0.3 mM was assayed at 307C by a filtration method (25) using 0.45-mm Nylaflo membrane filters (Gelman Sciences). Protein was estimated (21) using bovine serum albumin as a standard. For tetracycline transport, cultures were prepared in the same way except for the omission of EDTA from the second wash. The uptake of [7-3H]tetracycline (0.6 Ci/mmol) at a final concentration of 500 mM was assayed as above except for the use of GN-6 Metricel membrane filters (Gelman Sciences) in place of the Nylaflo filters used in the TPP/ assay. Each filter type was chosen on the basis of the lowest background of radioactivity caused by the nonspecific binding of radiolabeled drug. All experiments were performed three times on separate days.

RESULTS

Construction and analysis of pAMG6. pMDR2000XS contains a full-length cDNA for the MDR1 gene within a 4380-nucleotide fragment (17). This clone was derived from a cDNA library from the MDR human KB carcinoma mutant line KB-C2.5 (26). The presence of long 5* and 3* untranslated regions either side of the cDNA translational reading frame is due to the original cDNA construction (26), in which overlapping cDNA clones were fused to produce the large insert in pMDR2000XS. The MDR1 sequence (GenBank; Accession No. M14758) was searched for unique restriction sites either side of—but not within—the reading frame of the gene. Unique sites for BstUI (010) and PmeI (/4042) were located. The BstUI site is 10 nucleotides upstream of the ATG start codon (/1), and the PmeI site is 200 nucleotides downstream of the TGA stop codon (/3840). The 4052 nucleotide BstUI–PmeIdigested and blunt-ended fragment was recovered from an agarose gel. pPOW B2 (Fig. 1) was digested with NdeI and SalI and then treated with Klenow polymerase to fill in the recessed 3* termini of NdeI and SalI. This blunt-ended vector (4811 nucleotide pairs) was ligated to the blunt-ended BstUI–PmeI MDR1 cDNA fragment (4052 nucleotide pairs) to generate two recombinant plasmid types with the MDR1 fragment in either orientation. The orientation of the MDR1 cDNA fragment could be deduced from XhoI and EcoRI double digests that will produce 2.9- and 6.0-kb fragments for the reverse orientation of the insert, and 1.24- and 7.6kb fragments for the correct orientation. Two JM109 transformants yielded plasmid DNA restriction fragments of 2.9 and 6.0 kb, consistent with MDR1 inserted in reverse, and the plasmid in one of these clones was

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FIG. 1. Construction of the recombinant plasmid pAMG6 containing a full-length cDNA of the MDR1 gene. The expression vector pPOW B2 was modified by firstly removing the pelB signal sequence and blunt-ending the NdeI and SalI restriction sites. This vector was then ligated to the blunt-ended BstUI–PmeI cDNA MDR1 fragment from pMDR2000XS. E. coli JM109 transformants containing the cDNA in either orientation were obtained and confirmed by restriction enzyme digestion and Southern hybridization. Only the pAMG6 recombinant with the MDR1 gene in the forward orientation is shown, with the 5-prime ligation junction indicated by the arrow, and the start codon of the MDR1 gene shown in bold type at the bottom of the sequence.

designated pAMG5; and a single transformant produced the 1.2- and 7.6-kb fragments that indicated the correct orientation of MDR1 in this recombinant plasmid, designated pAMG6. The molecular cloning scheme for pAMG6, which shows the forward orientation of the cDNA insert only, is depicted in Fig. 1. Posi-

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tive hybridization signals of the MDR1 cDNA in both pAMG5 and pAMG6 were obtained by Southern hybridization of the blotted XhoI and EcoRI gel fragments obtained above, using the 3.0-kb EcoRI cDNA intragenic fragment from pMDR2000XS as probe (data not shown). In addition, DNA sequencing across the vector/

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FIG. 2. Growth curves for E. coli UT5600 cells containing pAMG6. Overnight LB broth cultures were diluted into fresh medium and grown with aeration at 307C to mid-log phase, then subdivided and induced at various temperatures. UT5600(pAMG6) grown at 307C (l) then moved to 377C (s), 38.57C (1), 407C (j), or 427C (n) or grown initially at 427C (h).

insert ligation junction in pAMG6 established that the BstUI end of the cDNA was indeed ligated to the bluntended NdeI–SacI site of the vector, indicated by the arrow near the depicted partial sequence of pAMG6 in Fig. 1; and with the ATG start codon indicated in bold type at the bottom of this sequence. The SD sequence used as the ribosome binding site could be either the GAGG 5-9 nt upstream of the MDR1 start codon and within the cloned cDNA sequence, or the vector’s SD sequence (GGAG) 17–21 nt further upstream. When the three transformants were plated onto LB agar containing ampicillin and incubated at the nonpermissive 427C, the two clones containing pAMG5 grew normally, but the clone containing pAMG6 failed to grow. A search of the MDR1 DNA sequence showed that there is no start codon near the vector/insert junction in pAMG5; and moreover, there are 10 nonsense codons within the first 120 nucleotides in all 3 translational frames for the reverse orientation of MDR1 in pAMG5. The presence of these nonsense codons would also eliminate downstream translational reinitiation (27) that might produce N-terminal truncated polypeptides. These observations suggest that pAMG5 is therefore an excellent control. In pAMG6, however, the potential for translational reinitiation is indicated by the existence of at least four in-frame reinitiation sites— with ATG codons and Shine–Dalgarno sequences–in the first 500 nucleotides of the sequence. Therefore, at this point it was difficult to determine if the apparent cell death of JM109(pAMG6) at 427C was due to the production or overproduction of full-length or truncated P-gp polypeptides. Growth rates. The observation made above that full or prolonged induction of pAMG6 at 427C might lead

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to cell death in the JM109 host prompted a closer appraisal of the growth of cultures containing pAMG6 at temperatures supporting partial to full induction, and in a more suitable host. E. coli UT5600 is an OmpT protease-deficient mutant that has been used for the expression of P-gp from the murine cDNA (14). Cultures of UT5600 containing either pAMG5 or pAMG6 were grown at 307C into early exponential phase and then were subdivided and transferred to higher temperatures, or were grown initially at 427C (Fig. 2). When UT5600(pAMG6) was transferred from 30 to 427C, growth slowed after a short equilibration period. At intermediate temperatures there were less pronounced effects on this slowing of growth for UT5600(pAMG6) (Fig. 2). When an attempt was made to commence growing a culture of UT5600(pAMG6) at 427C, there was no increase in absorbance (Fig. 2), indicating that at this nonpermissive temperature the full induction of a product from pAMG6 was possibly bactericidal, or at least bacteriostatic. In contrast, UT5600(pAMG5) continued to grow normally when transferred from 30 to 427C (data not shown). The results suggest that by moderating the induction temperature, it is possible to maintain the viability of the cells producing a putative P-gp molecule in UT5600 (pAMG6). Detection of P-glycoprotein in E. coli membranes. Pgp was detected in E. coli membranes by Western blotting using C219 monoclonal antibody. Immunoreactive bands representing P-gp were detected only in membranes prepared from UT5600(pAMG6) cells, induced at 427C for 10 (lane 1), 20 (lane 2), or 40 (lane 3) min, respectively (Fig. 3). The apparent size of P-gp was approximately 130 to 140 kDa. Membrane proteins from uninduced UT5600(pAMG6) cells (lane 4), or from UT5600 host cells only (lane 5), showed no immunoreactive bands in the position expected for P-gp. Minor bands occurred in all lanes at a lower molecular size,

FIG. 3. Detection of P-glycoprotein in E. coli membranes. Solubilized membrane proteins were subjected to electrophoresis in a 1.0% SDS–5.6% polyacrylamide gel containing 9 M urea, and immunoblotted with the C219 anti-P-gp monoclonal antibody. UT5600(pAMG6) cells grown at 307C to mid-log phase were induced at 427C for 10 (lane 1), 20 (lane 2), and 40 min (lane 3). Lanes 4 and 5 contain membrane proteins from uninduced UT5600(pAMG6) cells and UT5600 host cells, respectively. The positions of molecular weight markers are indicated on the left-hand side of the figure.

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FIG. 4. Resistance of E. coli UT5600 containing pAMG5 or pAMG6 to TPA/. Overnight LB broth cultures were diluted into fresh medium and grown at 307C until they reached an absorbance of 0.2 at 600 nm, transferred to 407C and induced for 30 min, then subdivided and their growth at 307C was monitored by absorbance at 600 nm, following the addition of given concentrations of TPA/. Symbols represent UT5600 harboring pAMG5 (l) or pAMG6 (s). A, B, C, and D represent relative growth rates versus drug concentration at 30, 60, 90, and 120 min, respectively. The drug IC50s are depicted by the dashed lines.

and these must represent native E. coli proteins that are immunoreactive to the C219 monoclonal antibody. Apart from these minor bands, there were no detectible proteolytic P-gp fragments. Drug challenge tests. P-gp substrates that are permeant to the EDTA-treated E. coli outer membrane include the lipophilic cations TPA/ and TPP/ (28). The protein synthesis inhibitor puromycin, which has been shown to be a competitive inhibitor of vinblastine transport in multidrug-resistant KB cells (5), is permeant to untreated E. coli cells. The effects of TPA/ (Fig. 4) and puromycin (Fig. 5) on cell growth were tested in liquid culture. Growth rates in the presence of different concentrations of either drug relative to growth in the absence of drug were consistently higher for UT5600(pAMG6) than for UT5600(pAMG5) cells. When growth at 307C was extended over a range of 30 to 120 min following the induction pulse at 407C, there was a reduction in the TPA/ IC50 for UT5600(pAMG6) from 7.9 mM after 60 min (Fig. 4B) to 3.4 mM after 120 min (Fig. 4D). The IC50s for the control UT5600(pAMG5) cells were lower than for cell harboring pAMG6 at all time points, but the differential between cells containing either plasmid became closer as the time of growth was extended. This suggests that the overexpression of product in UT5600(pAMG6) was beginning to have an effect on cell growth with time. Although the IC50 for

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UT5600(pAMG6) was not able to be measured at 30 min (Fig. 4A), the high relative growth rate (about 80% in 8 mM TPA/) showed that these cells are significantly more resistant than the control UT5600(pAMG5) cells at this shorter time interval. When UT5600 cells containing either pAMG5 or pAMG6 were challenged with increasing concentrations of puromycin (Fig. 5) following an induction pulse at 407C, the results obtained were similar to those for TPA/. UT5600(pAMG6) cells were more resistant than UT5600(pAMG5) control cells, even after 120 min of postinduction growth at 307C in the presence of drug (IC50 of 280 mM versus 85 mM, Fig. 5D). The IC50 for puromycin in the UT5600(pAMG5) control cells could be estimated at 60 min (Fig. 5B) but not before 120 min in the UT5600(pAMG6) test cells. These results suggest that the MDR1 cDNA gene produces a functional P-gp that confers in vivo resistance in E. coli to P-gp substrates. Similar results were obtained for TPP/ cytotoxicity assays with cells containing either plasmid (data not shown). Transport assays. The lipophilic cations TPA/ and TPP/ are P-gp substrates that are permeant to EDTAtreated E. coli cells (14, 28). Induced UT5600(pAMG6) cells energized with D-lactate showed a threefold lower accumulation of [3H]-TPP/ than either uninduced UT5600(pAMG6) or UT5600 host cells (Fig. 6). This reduced

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FIG. 5. Resistance of E. coli UT5600 containing pAMG5 or pAMG6 to puromycin. Overnight LB broth cultures were diluted into fresh medium and grown at 307C until they reached an absorbance of 0.2 at 600 nm, transferred to 407C and induced for 30 min, then subdivided and their growth at 307C was monitored by absorbance at 600 nm, following the addition of given concentrations of puromycin. Symbols represent UT5600 harboring pAMG5 (l) or pAMG6 (s). A, B, C, and D represent relative growth rates versus drug concentration at 30, 60, 90, and 120 min, respectively. The drug IC50s are depicted by the dashed lines.

accumulation was energy-dependent because the addition of the protonophore CCCP, which dissipates the energized membrane state (29), caused an increase in accumulation of [3H]TPP/ by induced cells and a corresponding decrease in uninduced or host cells (Fig. 6). In the absence of the added energy substrate D-lactate, the accumulation of TPP/ was higher in all three cell samples (data not shown). D-Glucose or reduced phenazine methosulfate could be substituted as alternative substrates to D-lactate with similar results (data not shown). In order to demonstrate that the reduced accumulation of TPP/ was not due to a loss of membrane potential, or to effects caused by the higher temperature used to induce the cells, L-[2,3-3H]proline accumulation was measured in uninduced and induced cells at both 30 and 427C, but the levels of uptake were similar (data not shown). Tetracycline is a weak substrate for P-gp (30), and this observation prompted an examination of tetracycline transport in E. coli cells harboring the cloned MDR1 gene. Although E. coli cells have an endogenous—albeit weak—tetracycline efflux system (25), this did not prevent the observation in this study of a noticeably reduced accumulation of [3H]tetracycline by induced, resistant UT5600(pAMG6) cells, compared to uninduced UT5600(pAMG6) cells, or UT5600 host cells (Fig. 7). This effect was only evident at a concentration

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of 500 mM tetracycline and not at the low concentrations (5 to 10 mM) of tetracycline used for detecting the endogenous bacterial efflux system (25). The rate of accumulation of tetracycline by all three cultures depicted different profiles to the TPP/ accumulation with an initial rapid uptake followed by a slower accumulation and equilibration (Fig. 7). This could either reflect the bacterial uptake/efflux and P-gp efflux systems working simultaneously, or perhaps a lower affinity of tetracycline for P-gp. DISCUSSION

The murine mdr3 isoform that confers an MDR phenotype and is marginally more closely related to the human MDR1 gene than is the murine mdr1 isoform has not yet been expressed in E. coli. Moreover, the murine and human isoforms elicit quantitative differences in their cross-resistance profiles (31), and it might therefore be of interest to examine the topological or functional nuances that produce different profiles in these isoforms. In this preliminary study, we report the cloning of the human MDR1 cDNA into a prokaryotic expression vector and the consequent functional expression of P-gp in E. coli. We have provided evidence of increased resistance to known P-gp substrates, namely TPA/, TPP/, and puromycin; of reduced accu-

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FIG. 6. Transport of [3H]TPP/ by E. coli UT5600 (j) and UT5600(pAMG6) uninduced (s) and induced at 427C (l). Cells were grown, induced, and treated with EDTA as described. Transport of 0.2 mM [3H]TPP/ in cells energized with 20 mM D-lactate was assayed at 307C by rapid filtration. CCCP (100 mM) was added at the points indicated by the arrows. The vertical bars are the standard deviations from three independent determinations.

mulation of TPP/ and tetracycline; and of the expression of a full-length immunoreactive P-gp in the membrane fraction of resistant cells. We have demonstrated that an E. coli host can indeed be employed as a model system for the study of the human P-gp molecule, and the potential for an analysis of the membrane topology and transport function of human P-gp has been established. Many eukaryotic genes have been expressed as heterologous proteins in E. coli, but others present difficulties that are related to size, copy number, stability, location, or function (32, 33). Although glycosylation of a protein may confer conformational stability and protease resistance (34), the unglycosylated P-gp molecule is known to be functional in eukaryotic cell tissue culture (6, 35). Thus, expression of the unglycosylated, heterologous protein in E. coli should not present problems of functionality. P-gp is a large polytopic membrane glycoprotein, and its expression in E. coli could depend upon the correct positioning of the MDR1 cDNA sequence in the expression vector, and the copy number and stability of the recombinant plasmid and mRNA transcripts. It was in consideration of these potential constraints that the molecular cloning strategy was devised to eliminate most of the noncoding sequence of the cDNA (Fig. 1). The transcriptional terminator on the vector increases the stability of the recombinant plasmid and the mRNA transcripts (18). In spite of these precautions, the fully induced expression of MDR1 in the unsuitable JM109 host resulted in cell

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death at 427C that was possibly caused by the overproduction of P-gp in an unsuitable host. The solutions to these problems were met by the choice of the OmpT protease-deficient UT5600 strain as host, following the success of its use for the expression of P-gp from the mouse mdr1 cDNA gene (14). The OmpT protease is associated with the outer membrane in E. coli, is specific for paired basic residues, and is inhibited by Zn2/ and Cu2/ (36). Degradation is believed to depend upon the paired basic residues and the overall conformation of the protein. Interestingly, in the predicted structural model for P-gp (4), there are 22 paired basic residues on the cytoplasmic side of the membrane, none within the transmembrane spans, and a single pair on the external side of the membrane in the C-terminal half of the protein. The demonstration of expression of P-gp under moderated induction (Fig. 2), and of in vivo resistance to TPA/ (Fig. 4) and puromycin (Fig. 5) exhibited by the UT5600(pAMG6) clone, suggests that UT5600 is a suitable host for the expression of human P-gp in E. coli. One of the major considerations in the attempt to express the MDR1 gene in E. coli was the potential problem of overproduction of P-gp and consequent toxicity. Such a concern is particularly relevant in view of the demonstrated toxicity to E. coli of the overexpressed tetA gene on Tn10 on high-copy-number plasmids, or from a derepressed, strong lPL promoter (37, 38). TetA is an integral inner-membrane protein that mediates resistance via an energy-dependent efflux of tetracycline (38), and its overproduction in E. coli results in cell death from the loss of membrane potential

FIG. 7. Transport of [3H]tetracycline by E. coli UT5600 (j) and UT5600(pAMG6) uninduced (s) and induced at 427C (l). Transport of 500 mM [3H]tetracycline in cells energized with 20 mM D-lactate was assayed at 307C by rapid filtration as described. The data points were averaged from three independent determinations.

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(37). The loss of membrane potential in cells that were induced to overproduce TetA protein was demonstrated by the lack of increased accumulation of [3H]TPP/ upon the addition of the uncoupler CCCP, and also by a severe decrease in the active uptake of proline (37). No such problem, however, occurred in this study after the induced expression of P-gp in UT5600 cells harboring pAMG6. The accumulation of [3H]TPP/ in these resistant cells was responsive to CCCP (Fig. 6), and the accumulation of proline by susceptible or resistant cells (uninduced and induced) was unchanged (data not shown), suggesting that the membrane potential component of the proton motive force was intact. Induced UT5600(pAMG6) cells also exhibited a reduced accumulation of [3H]tetracycline, which is in agreement with the demonstration elsewhere that tetracycline may be transported by P-gp in human MDR cells (30). P-gp was detected in immunoblots (Fig. 3) as a single molecular species of approximately 130 to 140 kDa, a size range that is consistent with the unglycosylated, intact molecule. Significantly, immunoreactive bands were only detected in membranes prepared from induced E. coli cells (Fig. 3), which supports the other data in this study for a membrane-located, intact, and functional human P-gp. Since the MDR1-encoded P-gp is functionally active in the cytoplasmic membrane of E. coli, a comparative study of the topology of this protein to the membrane topology of the mouse mdr1-encoded P-gp would be warranted. A major revision of the membrane topology of the mouse P-gp was suggested from the results of the study of alkaline phosphatase hybrids of the molecule (15, 16). This revision differs from the predicted structure of the human P-gp (4) by placing transmembrane hydrophobic domains in an aqueous environment, which can be explained by intramolecular hydrophobic interaction or by oligomerization (15). For the mammalian system, P-gp has been recognized as being inserted in membranes in different topological forms in terms of the orientation and oligomerization of the protein, and it may be that different conformations of the protein are associated with different functions (39–41). This might help to explain the anomaly of the diverse substrate range of P-glycoprotein that is unique to this member of the ATPase superfamily. The present study now provides an opportunity to examine the topology and other features of human P-gp in E. coli membranes. Some of the problems associated with the study of ATP-dependent P-gp-mediated transport may be amenable to an E. coli model in which the membrane-embedded human P-gp molecule is able to function as a transporter. Transport in E. coli has been extensively studied and parameters can be carefully arranged to account for the effects of substrates, inhibitors, and uncouplers (29). Although EDTA-treated E. coli cells are permeant to some drugs that cannot cross the outer

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membrane, there are mutants available that do not require such treatment (42), and the use of vesicles would not only overcome the permeation problem, but also allow the addition of reactants to either side of the P-gp molecule. This would allow the testing of several other P-gp substrates and inhibitors such as verapamil. An important follow-up study will also include at least one transport-defective P-gp with a missense mutation, that does not affect the protein’s localization in the membranes of animal cells (14). A recent report (43) in which highly purified P-gp was reconstituted in a liposome system in a uniformly inside-out orientation, and used to assay the transport of a fluorescent P-gp substrate, provides evidence that P-gp alone was sufficient to transport drugs out of the membrane bilayer. The evaluation of the mechanism of P-gp-mediated multidrug resistance has remained somewhat elusive and may remain so even in the E. coli system described in this study. However, the obvious structural and functional similarities of P-gp to prokaryotic ABC transporters and other efflux transporters argues for a more complete study of the consequences pertaining to the expression of human P-gp in E. coli. REFERENCES 1. Patel, N. M., and Rothenberg, M. L. (1994) Invest. New Drugs 12, 1–13. 2. Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583–2592. 3. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385–427. 4. Gottesman, M. M., and Pastan, I. (1988) J. Biol. Chem. 263, 12163–12166. 5. Horio, M., Gottesman, M. M., and Pastan, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3580–3584. 6. Endicott, J. A., and Ling, V. (1989) Annu. Rev. Biochem. 58, 137–171. 7. Ruetz, S., and Gros, P. (1994) Trends Pharmacol. Sci. 15, 260– 263. 8. Kuchler, K., and Thorner, J. (1992) Endocr. Rev. 13, 499–514. 9. Fath, M. J., and Kolter, R. (1993) Microbiol. Rev. 57, 995–1017. 10. Childs, S., and Ling, V. (1994) in Important Advances in Oncology (DeVita, V. T., Hellman, S., and Rosenberg, S. A., Eds.), pp. 21–36, Lippincott, Philadelphia, PA. 11. Simon, S. M., and Schindler, M. (1994) Proc. Natl. Acad. Sci. USA 91, 3497–3504. 12. Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, Uzi., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbarb, R. E., and Higgins, C. F. (1990) Nature 346, 362–365. 13. Doige, C. A., and Ames, G. F-L. (1993) Annu. Rev. Microbiol. 47, 291–319. 14. Bibi, E., Gros, P., and Kaback, H. R. (1993) Proc. Natl. Acad. Sci. USA 90, 9209–9213. 15. Bibi, E., and Beja, O. (1994) J. Biol. Chem. 269, 19910–19915. 16. Beja, O., and Bibi, E. (1995) J. Biol. Chem. 270, 12351–12354. 17. Pastan, I., Gottesman, M. M., Ueda, K., Lovelace, E., Rutherford, A. V., and Willingham, M. C. (1988) Proc. Natl. Acad. Sci. USA 85, 4486–4490.

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