THE JOURN*L OF BIOLCGICAL CHEMIYTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 31, Issue of August 5, pp. 19910-19915, 1994 Printed in U.S.A.

Membrane Topology of Multidrug Resistance Protein Expressed in Escherichia coli N-TERMINAL DOMAIN*

(Received for publication, April 18, 1994, and in revised form, May 27, 1994)

Eitan BibiS and Oded Beja From the Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Expression of eukaryotic polytopic membrane pro- teins in Escherichia coli could provide an invaluable system for structure-function studies. Recently, the functional expression of a mouse multidrug resistance protein (Mdrl) in E. coli was described (Bibi, E., Gros, P., and Kaback, H. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9209-9213). In the present study, the phoA gene fusion approach has been utilized to determine the membrane topology of the N-terminal domain of Mdr. The results support the idea that the N-terminal half of Mdr con- tains six transmembrane helices (TMs). However, our observations suggest that the previously proposed TM4 (amino acid residues ThP4-AlaZs2) is located at the periplasmic face of the membrane. Alternatively, we pro- pose that another stretch of amino acid residues (LeuMS (out) to Ile2” (in)) traverses the membrane. This assign- ment is indicated also by the following observations: 1) deletion of a segment containing the originally pre- dicted TM4 (AT214-K241) does not reverse the orienta- tion of the Mdr-alkaline phosphatase hybrid that is lo- cated in the following cytoplasmic loop; 2) a “sandwich” chimera, in which alkaline phosphatase is inserted in- frame between amino acid residues Leu226 and Sera2’, exhibits high alkaline phosphatase activity. The exist- ence of an externally exposed hydrophobic domain in Mdr may have special structural and functional impli- cations, and these may also be relevant to other mem- bers of the ABC superfamily.

The simultaneous emergence of resistance of cultured cells in vitro and tumor cells in viuo to many hydrophobic chemothera- peutic drugs is termed multidrug resistance (MDR)’ (Endicott and Ling, 1989). MDR is caused by the overexpression of a 170-kDa membrane protein called Mdr or P-glycoprotein (Gros and Buschman, 19931, which binds analogs of ATP (Schurr et al., 1989) and cytotoxic drugs (Safa et al., 1986b) and exhibits ATPase activity (Sarkadi et al., 1992). Although the ability of mdr genes to confer multidrug resistance directly has been established in transfection experiments (Gros et al., 1986b), the exact mechanism by which P-glycoprotein mediates drug efflux

* This research was supported in part by the MINERVA Foundation, Munich, Germany and by the Leo and Julia Forchheimer Center for Molecular Genetics, Weizmann Institute of Science. The costs of publi- cation of this article were defrayed in part by the payment of page

in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. charges. This article must therefore be hereby marked “aduertisement”

$ Incumbent of the Dr. Samuel 0. Freedman Career Development Chair in the Life Sciences. To whom correspondence should be ad- dressed: Fax: 972-834-4118.

The abbreviations used are: MDR, multidrug resistance; TM, trans- membrane helix; IPTG, isopropyl-l-thio-6-o-galactopyranoside; PCR, polymerase chain reaction.

remains obscure. In this regard, information on the membrane topology of the protein is essential.

A general secondary structure model for Mdr has been de- duced from hydropathy profiling (see Fig. lA for the N-terminal domain). I t is proposed that Mdr is a polytopic integral mem- brane protein composed of two similarly organized domains. Each domain contains six putative transmembrane helices (TMs) (see Fig. 1B for the N-terminal domain) and a large cytoplasmic nucleotide binding domain. The two halves of the molecule are connected by a highly charged segment termed a linker. The same general organization is proposed for other transport proteins belonging to the ABC (Hyde et al., 1990) or traffic ATPase (Mimura et al., 1991) superfamily. Major fea- tures of the orientation of Mdr have been elucidated using sequence-specific antibodies (Kartner et al., 1985; Yoshimura et al., 1989; Georges et al., 1993). More specifically, it is suggested that the Mdr molecule has 12 TMs with the C and the N termini, as well as both nucleotide binding domains, located on the cytoplasmic surface of the membrane (Gottesman and Pas- tan, 1988). Experimental confirmation of the predicted mem- brane topology would facilitate genetic and biochemical studies of the function of Mdr. However, recent studies have indicated that the topology of Mdr in the membrane may be different from the predicted structure (Zhang and Ling, 1991; Zhang et al., 1993; Skach et al., 1993).

Recently, we have shown that the mouse Mdrl protein can be expressed in Escherichia coli in a functional state and at a relatively high level (Bibi et al., 1993). One advantage of this heterologous expression system for eukaryotic integral mem- brane proteins is the possibility of applying well characterized genetic methods to analyze membrane protein topology in E. coli (reviewed by Traxler et al. (1993)). Briefly, gene fusions encoding hybrid proteins composed of N-terminal fragments of the membrane protein attached to a cytoplasmic or a periplas- mic reporter lacking its signal peptide are expressed in E. coli. A periplasmic reporter requires export to the periplasm in or- der to be enzymatically active and acts as a sensor for periplas- mic location of the protein sequence to which it is attached (Calamia and Manoil, 1992). We have chosen to use alkaline phosphatase as the periplasmic reporter (Manoil and Beck- with, 1985). In the following study, a detailed analysis of Mdr- alkaline phosphatase fusion proteins in the N-terminal half of Mdr is described.

Overall, the results suggest that there are six TMs in the N-terminal half of the Mdr. However, the location of TM4 is different from the location proposed in the model. We conclude that a stretch of hydrophobic amino acid residues (Thr214- Alaz3’) previously proposed to cross the membrane is probably located on the external surface of the membrane. The possible role of such a membrane-exposed hydrophobic domain in the assembly of membrane proteins and in their function is dis- cussed below.

19910

Membrane Topology of Mdr Expressed in E. coli 19911

B

FIG. 1. Hydropathy plot and the deduced secondary structure model of the N-terminal transmembrane domain of mouse Mdrl. A, the average local hydrophobicity at each residue calculated by the method of Kyte and Doolittle (19821 is plotted on the vertical axis uersus the residue number on the horizontal axis. Higher values represent greater hydrophobicity. The figure is adopted from the output of the program DNA Strider. B, secondary structure model according to Gottesman and Pastan (1988). The arrow connectingA and B indicates a hydrophobic segment that was not considered to be a TM.

EXPERIMENTAL PROCEDURES MateriaZs-5-Bromo-4-chloro-3-indolyl phosphate, p-nitrophenyl

phosphate, isopropyl-I-thio-P-D-galactopyranoside (IPTG), and protein A (cell suspension of Staphylococcus aureus Cowan strain) were from Sigma. Restriction enzymes were obtained from New England BioLabs, and modifying enzymes were obtained from Boehringer Mannheim. Oligodeoxynucleotides were synthesized on an Applied Biosystems DNA synthesizer.' [35S]Methionine was obtained from Amersham Corp., and antibodies to alkaline phosphatase were from 5 Prime + 3 Prime, Inc. Prestained protein molecular weight markers were purchased from Bio- Rad, and DNA molecular weight markers were purchased from Life Technologies, Inc. Geneclean Glassmilk DNA purification kits were ob- tained from Bio 101, and Magic Mini Prep kits were obtained from Promega. All other materials were reagent grade and obtained from commercial sources.

Bacterial Strains-E. coli CC181 (araD139 A(ara, leu)7697, AZacY X74, phoAA20, galE, galK, thi, rpsE, rpoB, argE(am1, recAI/F' lacZq lacY328(am) pro') was obtained from Colin Manoil and used for DNA manipulations and screening for phoA' phenotypes. E. coli UT5600 (omp2") was obtained from the E. coli Genetic Stock Center at Yale University (strain 7092) and used for the expression of the Mdr-alkaline phosphatase hybrid proteins.

Construction of phoA Fusions-Initially, a general vector was con- structed in which phoA was fused with mdrl. Using polymerase chain reaction (PCR), an NheI site was created at the 3' end of mdrl in plasmid pT7-5/mdrl (Bibi et al., 1993). ThephoA gene was then isolated from pT7-5lZacYlphoA (Bibi et al., 1991) by NheI-Hind111 restriction and ligated with pT7-5/mdrl digested with the same enzymes. The final construct (pTmp) encodes full-length Mdrl with alkaline phosphatase attached in-frame to its C terminus (termed hybrid S1276). The PCR fragment used to create the NheI site (PstI-NheI in pTmp) was se- quenced, as well as the mdrl/phoA joint, to confirm that the frame was not changed. pTmp was then used to create all the other fusions by PCR as follows. Synthetic antisense oligodeoxynucleotides' were made for each chosen fusion joint. Each of them was constructed with a hanging tail containing an NheI site in-frame with the codon to which we planned to fuse phoA. Sense oligodeoxynucleotides were also synthe- sized, overlapping mdrl sequences with already existing unique sites. Using pairs of oligodeoxynucleotides (sense and antisense for each fu- sion) and a template (pT7-5/mdrl), DNA fragments were amplified by PCR. Every PCR product was isolated from 1.2% agarose gel and di- gested with the appropriate restriction enzymes. One enzyme recog- nizes the unique site constructed at the 5' end and NheI at the 3' end. After restriction, the fragment was purified again from 1.2% agarose and ligated with the appropriate fragment isolated from pTmp digested with the same two enzymes. The resulting vector encodes alkaline phos- phatase devoid of the signal peptide coding sequence but connected

The complete list of the synthetic oligodeoxynucleotides used in this study is available upon request.

in-frame with the appropriate N-terminal portion of the Mdr protein. The new plasmid was named aRer the most C-terminal Mdrl amino acid residue, which is located at the fusion joint. All of the plasmids were sequenced through the PCR-amplified region and the fusion joint. To delete the hydrophobic domain ( L e ~ ~ ' ~ - L y s ~ ~ ~ ) from hybrid S297, a sense deoxyoligonucleotide primer was synthesized. The primer con- tains the sequence encoding amino acids G 1 ~ ~ ~ ~ - A l a ~ ~ ~ and an NheI site in its 5' end. This primer and an antisense primer that was made for the construction of hybrid S297 were used to amplify the coding sequence for G ~ u ~ ~ ~ - S ~ ? ~ ~ withNheI sites in both ends by PCR. The purified DNA fragment was digested with NheI and ligated with the vector encoding hybrid L213 linearized withNheI. The new construct (S297A(214-241)) was sequenced through the PCR-amplified region and the NheI joints. The mutations that were caused by the manipulations are as follows: in hybrid G287, AsnZe6 was replaced by methionine, by leucine, and

position 288. In hybrid S297A(214-241) an insertion ofAla214 and Ser215 GlyZa7 by alanine, and in addition, a leucine residue was inserted at

was caused by the manipulation. In order to prepare the "sandwich hybrid (Ehrmann et al., 1990), the phoA gene was amplified by PCR using sense and antisense deoxyoligonucleotides and using the full- length mdrllphoA fusion vector as a template. The resulting 1.3-kilo- base fragment was then isolated, treated with T4 DNA polymerase and Klenow fragment, phosphorylated with T4 DNA kinase (to create the phoA cassette gene), and ligated to alkaline phosphatase-treated pT7- 5/mdrl linearized by Mum1 (Boehringer Mannheim). DNApreparations from blue colonies produced by E. coli UT5600 (ompT-) transformed with the ligation mixture were analyzed by restriction enzymes and sequenced through the two mdrllphoA joints.

Alkaline Phosphatase Activity Analysis-Overnight cultures of E. coli (CC18UphoA') or UT5600(ompT-)) cells harboring the mdr-phoA gene fusion or the plain vector as negative control were diluted 1 5 0 into 2 ml of LB containing 100 pg/ml ampicillin and grown for 3 h at 30 "C. The cells were then induced with IPTG (1 mM final concentration) for 2 h at 30 "C. 1 ml of each culture was then centrifuged and washed once with 1 ml of ice-cold 10 mM Tris-HC1, pH 8, 10 mM MgSO,, and 1 mM iodoacetamide. Pellets were suspended in ice-cold 1 M Tris-HC1, pH 8, and A,,, of the diluted cells (1: lO) was measured. 0.1 ml of cells were then transferred into 0.9 ml of 1 M Tris-HC1, pH 8, 0.1 mM ZnCl,, and 1 mM iodoacetamide. One tube without cells was used for control. 50 pl of 0.1% SDS was then added, and the tubes were vortexed and incubated a t 37 "C for 5 min to permeabilize the cells. The samples were placed on ice for 5 min, mixed with 0.1 ml of 0.4% p-nitrophenyl phosphate (in 1 M Tris-HC1, pH 81, and heated to 37 "C. When a pale yellow color ap- peared, the time was noted, 120 p1 of 0.1 M EDTA, pH 8, 1 M GHPO, were added, and the tubes were placed on ice to stop the reaction. Cells were pelleted, and of the supernatant was measured. Alkaline phosphatase activity was calculated as suggested by Brickman and Beckwith (1975).

[35SlMethionine Labeling and Immunoprecipitation-E. coli cells harboring the fusion vectors or pT7-5 for control were grown overnight at 30 "C in M9 minimal media containing 0.5% glycerol, 100 pg/ml ampicillin, 1 pg/ml thiamine, and all of the amino acids (15 pglml) except for methionine and cysteine. Cells were diluted 1:IO and grown for 4 h a t 30 "C. IPTG (0.5 mM final concentration) was then added for 30 min. 0.6 ml of the cultures (A4', = 0.5) were transferred into pre- warmed (30 "C) tubes containing 10 pCi of [36Slmethionine (1000 Ci/ mmol) and incubated a t 30 "C for 5 min. Labeled cells (0.5 ml) were treated and immunoreacted with antialkaline phosphatase antibodies as described (Manoil, 1991). Immunoprecipitated material was ex+ tracted in 50 p1 of sample buffer (Laemmli, 1970) and separated on SDS-polyacrylamide gel electrophoresis, and the dry gel was exposed to film for at least 24 h. Radioactive bands were quantitated by a densi- tometer. Prestained molecular weight markers were used to estimate the molecular weight of the different hybrid proteins.

RESULTS

The study was performed in four stages. Initially, we ana- lyzed 10 Mdr-alkaline phosphatase hybrids in each predicted hydrophilic loop in the N-terminal half of Mdr. The results obtained from this set of fusions revealed a problematic region between putative TM3 and TM5. Therefore, a second series of fusions were constructed in order to map the topology of the region in question. To test the results obtained with hybrid Mdr-alkaline phosphatase proteins, a previously proposed TM was deleted, and the effect of the deletion on the topology was studied. In addition, we utilized the "gene sandwich" approach

19912 Membrane Topology of Mdr Expressed in E. coli

400 n f = i l

FUSION

B " - 80

80 - 49.5"- - 4 9 . 5

CC181 UT5600

FIG. 2. Comparison between E. coli strains CClBl (phoA-) and UT5600(ompT").A, alkaline phosphatase activity exhibited by various Mdr-alkaline phosphatase hybrids expressed in each strain. B, immu- noprecipitation of I:"Slmethionine-labeled CC181 (12% gel) or UT5600 (10% gel) cells expressing various Mdr-alkaline phosphatase hybrids. BZack dots represent the proposed full-length hybrids.

(Ehrmann et al., 19901, in which phoA was inserted in-frame into a specific site.

In general, the experiment was designed so tha t at least one fusion in each loop was placed in the C-terminal portion of the loop. This design should prevent the disruption of known top- ological determinants, such as positively charged residues, nor- mally located in the N-terminal region of the loops (Boyd et al., 1993). All of the other fusions were directed to chosen sites. A vector template was made (see "Experimental Procedures") in which the phoA gene (devoid of the leader peptide coding se- quence) was fused to the 3' end of mdrl wi th a unique NheI restriction site in the junction. Using synthetic deoxyoligo- nucleotides for polymerase chain reaction, given fusions were constructed. Each fusion was made by PCR amplification of a 5' region of the mdr gene encoding the N-terminal Mdr polypep- tide with the appropriate amino acid residue on its C terminus. The PCR-amplified fragments were designed to contain an NheI site in the 3' end and another unique site in the 5' end. After digestion with these two restriction enzymes, fragments were ligated with the vector template digested with the same two enzymes. Positive transformants were detected by alkaline phosphatase activity on 5-bromo-4-chloro-3-indolyl phosphate plates, and the DNA was analyzed by restriction enzyme diges- tion and by sequencing of the PCR-amplified region and the NheI junction. The screening was made with an E. coli strain devoid of the phoA gene (CC181). Therefore, constructs with a very low alkaline phosphatase activity could also be detected on the negative background.

The first series of hybrids was analyzed initially in E. coli CC181. Alkaline phosphatase activity was measured and sug- gested a six-TM organization for the N-terminal half of the protein (Fig. 2 A ) . However, all of the attempts to detect full- length ["Slmethionine-labeled hybrid proteins were unsuccess- ful. Previously, we have observed that E. coli UT5600 (ompT-) is able to express Mdr in a relatively stable state (Bibi et al., 1993). Therefore, we have repeated the analyses of the first series of fusions with the ompT- strain. As expected, the abso- lute alkaline phosphatase activity of hybrids expressed by this strain was about 5 times higher than by CC181 (Fig. 2 A ) . However, the profile of the activities of fusions along the polypeptide chain was remarkably similar for both strains.

A comparative immunoprecipitation experiment in which UT5600 and CC181 cells expressing various hybrid proteins were labeled with ["Slmethionine is shown in Fig. 2B. Using antialkaline phosphatase antibodies, the immunoprecipitated material was separated by gel electrophoresis, and the results demonstrate that full-length hybrids are expressed at a much higher level in UT5600 than in CC181 but still exhibit proteo- lytic products. In order to use E. coli UT5600 (which is phoA') in alkaline phosphatase activity assays, its basal activity was determined (data not shown). Under regular growth conditions in LB broth, the genomic alkaline phosphatase is not induced, and the basal activity observed is extremely low even if com- pared with that of an inactive cytoplasm-oriented alkaline phosphatase chimera. Consequently, we used E. coli UT5600 strain for the remainder of the experiments. The first set of fusions was then analyzed for the expression of the resulting hybrids and their alkaline phosphatase activity. The expression level was measured by labeling with ["Slmethionine and im- munoprecipitation by antialkaline phosphatase antibodies (Fig. 3A 1. As shown, the level of expression is gradually reduced with the size of the hybrid proteins, although the number of methionine residues increases. This may be attributed either to decreased expression or to the instability of longer polypep- tides. The proposed full-length hybrids shown in Fig. 3 are indicated by black dots. However, a few deviations from the expected increase in the molecular weight between sequential hybrids are observed (for example the band representing hy- brid E109 in Fig. 3A is higher than that of hybrid R147). This is probably caused by the addition of a hydrophobic segment (for example TM2 in hybrid R147), and i t is known that hydro- phobic integral membrane proteins can exhibit high mobility in SDS gels.

Many of the Mdr-alkaline phosphatase hybrids exhibit major proteolytic fragments that are immunoprecipitated with anti- alkaline phosphatase antibodies (see for example Fig. 3A). Since it is possible that a certain proteolytic product exhibits alkaline phosphatase activity as well as its precursor (the ap- propriate full-length hybrid) it was also considered for the cal- culation of the normalized alkaline phosphatase activities. By use of a densitometer, the bands representing the expected full-length chimeric proteins or the full-length hybrids com- bined with the appropriate major proteolytic products were quantitated. The relative observed expression pattern was found to be similar with or without the proteolytic fragments. Therefore, only the densitometry of the full-length hybrids was considered. In addition, the number of methionine residues was taken into account because I"S1methionine was used for label- ing. For example, the largest hybrid protein (1374) contains 17 methionine residues, whereas the shortest hybrid protein (K47) contains only 10. The level of expression (band intensity) di- vided by the number of methionines was then used to normal- ize the alkaline phosphatase activity, as presented in Table I (experiment a). Fusions K47 and K80 exhibit low ( 5 ) and high (64) alkaline phosphatase activity, respectively, and therefore we suggest that they flank TM1. Similarly, hybrids E109 and R147 (high and low activity, respectively) are consistent with the secondary structure model proposed for TM2, and the low activity of L170 with the high activity of L213 identifies the borders of TM3. Surprisingly, however, fusion Y246 exhibits high alkaline phosphatase activity despite its predicted loca- tion in the cytoplasm, whereas the following fusion G287 shows low activity, as expected from the predicted orientation. TM5 is probably located between amino acid residues GlyZxi and ValT3" (low and high activities, respectively), as predicted. The last TM of the N-terminal half of Mdr, TM6, also behaves a s ex- pected, flanked by amino acid residues Val"" and Ile"'. Fusions

Membrane Topology of Mdr Expressed in E. coli 19913

A

B

C

Kd

80 -

4 9 . 5 -

Kd

106-

80 -

49.5-

Kd

106-

80 -

49.5-

1 2

m w I N

P N H c l a

1 2 3 4 5 6 7 8 9 1 0 1 1 Normalized alkaline Phosphatase activity of various hybrids T:WI.K I

Normalized activity = (activity x number of methionines)/(expression level x 10).

1 2 3 4 5 6 1 8 9 ~

D

3 4 Kd

a

1

205

2 3

W P N rl UI

W N N cl c m UI

m I P B a

FIG. 3. Immunoprecipitation of [S5S]methionine-labeled UT- 5600 cells expressing various Mdr-alkaline phosphatase hy- brids. A and R , 10% gel; C and D, 8 9 gel. Black dots represent the proposed full-length hybrids.

a t these residues (Valssn and IIe3T exhibit high and low alka- line phosphatase activities, respectively.

The deviation of hybrid Y246 from the predicted orientation of TM4 suggested, therefore, a detailed topological study of the previously proposed cytoplasmic loop connecting TM4 and TM5. To this end, we constructed seven Mdr-alkaline phospha- tase hybrids and used immunoprecipitation of ["Slmethionine- labeled hybrids for quantitative evaluation of their expression level (Fig. 3B ). Consequently, the alkaline phosphatase activi- ties of the new hybrids were assayed, and the calculated nor- malized activities are presented in Table I (experiment b). The

Fusion Nu:her p,$:!$2:&e Expression lrvel Normalized activity methionines actwity

Exp. a K47 K80 E109 R147 L170 L2 13 Y246 G287 V330 I374

Exp. b E109 S179 T236 A259 G268 Y276 S297 V320

Exp. c L226 S297 S297A(214 SanL226 S1276

10 13 13 14 15 16 16 16 16 16

13 15 16 16 16 16 16 16

16 16

-241) 16 37 37

uni/s

22 344 506

34 70

303 211

7 365

59

506 20

187 64 33 30 34

169

146 34 41 35

126

pisrls I1 000

4.8 7 8.5 4.7 4.4 3.1 2.6 2.7 2.5 2.4

8.5 3.9 4 4.6 2.3 4.8 2.2 0.6

2.3 2.2 2.5 1

11.7

5 64 77 10 24

157 130

4 233

39

77 8

75 22 23 10

433 25

102 25 26

130 40

results obtained with hybrid S179 that exhibits low alkaline phosphatase activity indicate that it is located in the cytoplas- mic face of the membrane, thus preceding TM3 (see also R147 and L170 in Table I (experiment a)). The results obtained with hybrids T236 (high activity) and "59 , G268, Y276, and S297 (low level of alkaline phosphatase activity) further suggest that TM4 is flanked by amino acid residues Leu'":' (out) and IIezfi" (in) as illustrated in Fig. 4. This region of the protein is hydro- phobic and indicated by an arrow connecting A and B of Fig. 1. To test if this domain ( L e ~ ' " ~ - I l e ~ ~ ~ ) is solely responsible for the cytoplasmic orientation of the following loop, a deletion mutant was made (hybrid 8297A(214-241)). The hybrid is composed of the cytoplasm-oriented fusion S297, which was deleted of the originally proposed TM4 (Fig. 5). The expression level and the alkaline phosphatase activity of hybrid S297A(213-241) were analyzed and compared with those of hybrids S297 and L226 (L226 was specially constructed as a positive control for this set of experiments). As presented in Fig. 3C and Table I (experi- ment c), the three hybrids are expressed to a similar level, but only L226 exhibits a high level of alkaline phosphatase activity, supporting the proposal that this domain is external. The al- kaline phosphatase activities of hybrid S297 and its deletion mutant S297A(214-241) are low and remarkably similar, fur- ther suggesting that the hydrophobic stretch of amino acid residues Leu2""-Ile2fin traverses the membrane (Fig. 5 B ) and that the deleted hydrophobic region containing amino acid resi- dues Thr2'"-Ala2s' (previously proposed TM4) does not affect the topology of the following cytoplasmic loop.

Although the gene fusion approach is the predominant method used to decipher the topology of prokaryotic membrane proteins (Traxler et al., 1993) it has one critical disadvantage. There are indications that in a few cases, C-terminal domains of membrane proteins are required to maintain the correct assembly and therefore the topology of N-terminal segments (Calamia and Manoil, 1990; Calamia and Manoil, 1992). TO avoid such artifactual results, another methodology has been developed by Ehrman et al. (1990), who have shown that it is

19914 Membrane Topology of Mdr Expressed in E. coli

FIG. 4. Secondary structure of T M s 3,4, and 6 of Mdr as pro- posed in this study. The single letter amino acid code is used, and hydrophobic TMs are shown in vertical boxes. The horizontal box indi- cates TM4 in the original secondary structure model. Mdr-alkaline phosphatase hybrids are indicated. Positively charged residues (K and R ) are strongly shaded, and negatively charged residues (E and D ) are slightly shaded.

\ 52 97

5297 FIG. 5. Illustration of the possible effect of a deletion ( T h P 4 -

LysZ4') on the orientation of hybrid 5297. A, in the original second- ary structure model; B , in the secondary structure model as proposed in this study. White circles, low activity; Black circles, high activity.

possible to insert the full-length alkaline phosphatase into ex- ternal or internal domains of the integral membrane protein MalF. Although it has not yet been determined whether the new approach will provide correct information in cases where such controversial observations were made with phoA fusions (Calamia and Manoil, 1990; Allard and Bertrand, 19921, it is believed that by this method the membrane protein remains intact and contains the required folding information. In an effort to avoid structural effects mediated by the lack of Mdr domains C-terminal to the fusion site, especially in the prob-

lematic region between TM3 and TM5, we have constructed a sandwich hybrid. In this construct (SanL226), the phoA gene was inserted in-frame into the Mum1 site between codons L226 and S227 located at the C-terminal part of the original putative TM4. A full-length Mdr-alkaline phosphatase chimera (S1276) was used as a control in the expression experiments and in the alkaline phosphatase activity assays. As shown in Fig. 30, the sandwich hybrid (SanL226) is not expressed very well in com- parison with the full-length hybrid (S1276), and its gel mobility is slower than that of hybrid S1276. However, the calculated normalized activity of the sandwich is relatively high (Table I (experiment c)), indicating that alkaline phosphatase in the sandwich is translocated to the periplasmic space. As was pointed out previously (Ehrmann et al . , 1990), the activity of periplasm-oriented alkaline phosphatase attached to another protein in both the N- and the C-termini is relatively low when compared to the regular cytoplasm-oriented phoA fusions. Therefore, because of the relatively high alkaline phosphatase activity exhibited by SanL226, we conclude that the region flanking the inserted alkaline phosphatase in the sandwich is external. These results support the contention that the location of the original putative TM4 is not inside the membrane but in the periplasmic space.

DISCUSSION The study of the topological organization of polytopic mem-

brane proteins of eukaryotic origin in the homologous system involves complex and limited techniques. Among these, the use of specific antibodies and proteolytic digestion have been proved to be informative but restricted because many regions in the protein are not antigenic or are not exposed. Recently, the use of an in v i tro translation system with microsomal mem- branes has become an important tool in studying membrane topology. However, it has not yet been confirmed that the i n vitro system represents the real in v ivo situation. In contradis- tinction, it has been shown that in v i tro synthesized proteins undergo different sorts of processing such as glycosylation (compare for example the work presented by Schinkel et al. (1993) with that of Zhang and Ling (1991)). In this article we describe the use of alkaline phosphatase fusions to study Mdr topology in E. coli.

The predicted secondary structure model of Mdr contains six TMs in the first half of the protein (Fig. 6A). The first two TMs are connected by a heavily N-linked glycosylated hydrophilic loop recently shown to be the only glycosylated domain in hu- man Mdrl (Schinkel et al., 1993). Our results not only support the predicted orientation of TM1 and TM2 but also suggest that glycosylation is not essential for the correct assembly of this domain because Mdr is not glycosylated in E. coli. This is not surprising, however, because it has already been proposed that glycosylation is not obligatory for Mdr function (Raymond et al., 1992; Schinkel et al., 1993; Bibi et al . , 1993). This topology of TM1 and TM2 is also consistent with other observations ob- tained with the murine Mdrl (Zhang et al . , 1993) and with the human Mdrl (Skach and Lingappa, 1993). The orientation of TM3, as reflected by fusions S179 and L213, supports the sec- ondary structure model. The alkaline phosphatase activity of hybrid L213 cIearIy suggests its extracellular Iocation, unIike the results obtained by Zhang et al. (19931, where almost equal distribution on both sides of the membrane was observed with a similarly constructed hybrid protein. On the other hand, we find the similarity between our results obtained from the study of the orientation of TM4 and the results obtained with the murine Mdrl (Zhang et al., 1993) most interesting. Our con- clusion from a detailed phoA fusion analysis of the region con- taining amino acid residues L e ~ ~ ~ ~ - G l y ~ ~ ~ is that TM4 in the original secondary structure model is translocated to the

Membrane Topology of Mdr Expressed in E. coli 19915

Mdr. Such a hydrophobic domain may also have a functional role in binding Mdr substrates that are primarily hydrophobic. These results may be related to other members of the ABC superfamily with similar hydrophobicity patterns (Manavalan et al., 1993). We would also like to mention that the location of

A

, COOH

B

FIG. 6. Alignment of the alkaline phosphatase activity of the various Mdr-alkaline phosphatase hybrids with the secondary structure models suggested by Gottesman and Pastan (1988) (A) and in this study (B) . White circles, low activity; Black circles, high activity.

periplasmic space, and part of the following hydrophobic stretch crosses the membrane (Fig. 6B). More specifically, in our study we demonstrated that hybrid Y246 exhibits high alkaline phosphatase activity and thus propose that it is translocated as is also suggested by Zhang et al. (1993), who demonstrated that 80% of a similar construct, pGPGP-N4, is found in the lumen of endoplasmic reticulum membranes. Moreover, when the reporter protein (pGPGP-N4C in Zhang et al. (1993)) was fused to the C-terminal end of the loop connecting the originally predicted TM4 and TM5, only 40% of the hybrid protein crossed the membrane, while the majority (60%) remained in the cytoplasmic space. In a similar manner, we show that a hybrid of alkaline phosphatase fused to the same cytoplasmic domain (G287 or S297) remains inactive on the cytoplasmic side of the membrane.

It is not the first time that a hydrophobic stretch of amino acid residues sufficiently long to cross the membrane has been proposed to reside in the aqueous environment (see for example the topology of the cytochrome b subunit of the bc, complex from Rhodobacter sphaeroides (Yun et al., 1991)). If such a domain is really exposed to the hydrophilic environment, it raises energetics problems that could be solved by intramolecu- lar hydrophobic interaction or by oligomerization. So far, both possibilities are plausible. On one hand, based on electron in- activation analyses (Boscoboinik et al., 1990), it has been pro- posed that Mdr exists as a homodimer in membranes of mul- tidrug-resistant cells. On the other, it is also conceivable that exposed hydrophobic domains in either half of Mdr interact with each other, thus stabilizing the spatial conformation of

TM4, as proposed in this study, has already been predicted by Gros et al. (1986a, 1988).

Acknowledgments-We are grateful to H. R. Kaback for helpful dis- cussions during this work and comments on the manuscript and to P. Gros for encouragement. We thank K. Stemple for synthesizing many of the primers used in this work.

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