©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multidrug Resistance Protein (Mdr)-alkaline Phosphatase Hybrids in Escherichia coli Suggest a Major Revision in the Topology of the C-terminal Half of Mdr (*)

Oded Béj , Eitan Bibi (§)

From the (1) Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent studies reveal that the organization of the multidrug resistance protein (Mdr) in the membrane is probably not exactly as predicted from hydropathy profiling. When expressed in Escherichia coli, phoA gene fusions can be utilized to study the membrane topology of Mdr. Using this approach, it was proposed recently that the N-terminal hydrophobic domain of Mdr spans the membrane six times, in a different fashion from that predicted by hydropathy analysis (Bibi, E. and Béj, O. (1994) J. Biol. Chem. 269, 19910-19915). In this study, we analyze [Abstract] mdr-phoA fusions constructed in the C-terminal half of Mdr. Overall, the results presented here lead to a significant revision in the membrane topology model of the C-terminal half of Mdr. The new topology is discussed with regard to the hydropathy profiles of the well characterized ABC proteins MalG and MalF, which are strikingly similar to those of the N- and C-terminal halves of Mdr, respectively.


INTRODUCTION

Multidrug resistance protein (Mdr)() is a complex integral membrane protein which plays a major role in drug resistance of tumors (reviewed by Endicott and Ling(1989); Gottesman and Pastan, 1993; Gros and Buschman, 1993). Mdr is a 170-kDa membrane protein which belongs to the ABC (Hyde et al., 1990) or traffic ATPase (Mimura et al., 1991) superfamily of transport proteins. Proteins in this family contain two homologous nucleotide binding domains and two integral membrane domains each containing six putative transmembrane segments in -helical conformation. Included are the cystic fibrosis transmembrane conductance regulator (Riordan et al., 1989), the yeast mating factor exporter (Ste6) (Kuchler et al., 1989; McGrath and Varshavsky, 1989), the hemolysin exporter (HlyB) (Felmlee et al., 1985), and many so-called periplasmic permeases in Gram-negative bacteria, such as the maltose transport system in Escherichia coli (Nikaido, 1994).

A general secondary structure model for Mdr has been deduced from hydropathy analysis. Presumably, Mdr contains 12 transmembrane segments (TM) with the C and the N termini, as well as both nucleotide binding domains, on the cytoplasmic surface of the membrane (Gottesman and Pastan, 1988). The two halves of the molecule are connected by a highly charged linker. General features regarding the orientation of Mdr have been elucidated using sequence-specific antibodies (Kartner et al., 1985, Yoshimura et al., 1989), but the detailed topology of the protein in the membrane is still elusive. Experimental analysis of the predicted membrane topology is important in order to facilitate genetic and biochemical studies of Mdr function. In this regard, recent studies (Zhang and Ling, 1991; Zhang et al., 1993; Skach et al., 1993, Bibi and Béj, 1994) indicate that the topology of Mdr in the membrane may differ from the predicted structure.

In previous studies, it was shown that mouse Mdr1 (Gros et al., 1986) can be expressed in E. coli in a functional state and at a relatively high level (Bibi et al., 1993). This opened the possibility of utilizing the well characterized prokaryotic gene fusion approach (reviewed by Traxler et al. (1993)) to study the topology of Mdr. In a recent report (Bibi and Béj, 1994) the heterologous expression system was used to analyze the topology of the N-terminal hydrophobic domain of Mdr by gene fusions with alkaline phosphatase as a reporter (Manoil and Beckwith, 1985). In this study, we present an analysis of Mdr-alkaline phosphatase fusions constructed in the C-terminal half of Mdr. The results suggest that the homologous halves of Mdr are asymmetric in the membrane.


RESULTS AND DISCUSSION

A major deviation from the predicted structure of Mdr has been readily observed when reporter polypeptides fused to the putative extracellular loop following TM7 fail to cross the membrane (Skach et al., 1993). Using the C-terminal 142-amino acid residues of bovine prolactin as a reporter in Xenopus oocytes or in in vitro expression systems, Skach et al.(1993) constructed and analyzed Mdr-prolactin hybrids in which the prolactin derived reporter was attached to amino acid residues 733 and 750, both located in the C-terminal half of human MDR1 (see Fig. 5A for the specific location of the fusions in the original topology model). We constructed Mdr-alkaline phosphatase hybrids at Val and Cys, with the reporter following putative TM7 and two more hybrids at Thr and Lys, where the alkaline phosphatase moiety is attached to residues preceding TM7. In the E. coli system, the intracellular orientation of the hybrids is judged by the fact that all of them exhibit low specific alkaline phosphatase activities that are estimated from the absolute activity and the level of expression as detected by immunoprecipitation using antibodies against alkaline phosphatase (Fig. 1A). The low activities indicate cytoplasmic disposition of the fused reporters, despite the existence of a relatively long hydrophobic amino acid residues stretch between Lys and Val (proposed TM7). Interestingly, in both the prokaryotic and the eukaryotic systems, the reporters fail to cross the membrane, even though they are located in the putative extracellular loop following TM7. Skach et al. (1993) suggested that TM7 by itself is not able to cross the membrane and requires topological information located in the following transmembrane segments. However, careful examination of the hydropathy profile of Mdr in this region (Fig. 2A) reveals a stretch of relatively hydrophilic amino acid residues in the middle of TM7 and raises the possibility that TM7 (Fig. 2B) could cross the membrane twice (Fig. 2C), with a short extracellular loop (composed of Asn, Gly, Cys, see Fig. 5B) between the two membrane-spanning segments. To study this possibility, we constructed additional hybrids, at Gly and Pro which are located in the vicinity of the short hydrophilic segment and at Leu which is located in the N-terminal portion of TM7. The activities and the immunoprecipitation analysis of this group of hybrids are presented in Fig. 1B and demonstrate that hybrid Leu (low alkaline phosphatase activity) and hybrids Gly or Pro (high alkaline phosphatase activity) are potential borders for the first transmembrane segment. Consequently, the second half of the original TM7 also crosses the membrane as indicated by the flanking active (Pro) and non active (Val and Cys) hybrids (Fig. 1B). Therefore, the results obtained with this set of fusions fit the new proposed arrangement of Mdr.


Figure 5: Secondary structure of the region containing TMs 7, 8, and 9 of Mdr as proposed in this study (B) and a previous model (A). The single amino acid code is used; hydrophobic TMs are shown in vertical boxes. Mdr-alkaline phosphatase hybrids are indicated. Positively and negatively charged residues are slightly shaded.




Figure 1: Right panels, immunoprecipitation with anti-alkaline phosphatase antibodies of [S]methionine-labeled UT5600 cells expressing various Mdr-alkaline phosphatase hybrids. Left panels, specific activities measured and calculated for the same Mdr-alkaline phosphatase hybrids as in the right panels. The same transformants were used for immunoprecipitation and alkaline phosphatase activity assays in each set (A, B, C, D). All the methods used for the preparation of the gene fusion constructs (the complete list of the synthetic oligodeoxynucleotides used in this study is available upon request), the immunoprecipitation, and the alkaline phosphatase activity assays were described in detail in Bibi and Béj (1994).




Figure 2: Hydropathy plot and the deduced secondary structure model of the C-terminal transmembrane domain of mouse Mdr1. A, the average local hydrophobicity at each residue calculated by the method of Kyte and Doolittle (1982) is plotted on the vertical axis versus 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). C, secondary structure model proposed in this study. The arrow indicates the orientation of the TM (N to C terminus)



To further examine the results, we constructed a ``sandwich hybrid'' (Ehrmann et al., 1990), SanV717, between amino acid residues Val and Asn (Ile was deleted due to the gene manipulation). The alkaline phosphatase inserted at this point is expected to be extracellular (Fig. 5B) if the insertion does not damage the structure of Mdr. However, as shown in Fig. 1D, the normalized activity of SanV717 is low relative to a previously described sandwich fusion that was constructed in an extracellular loop but in the N-terminal half of Mdr (SanL226, Bibi and Béj, 1994). As expected, full-length Mdr-alkaline phosphatase hybrid Ser also exhibits low alkaline phosphatase activity (Fig. 1D) consistent with its cytoplasmic orientation. These observations thus indicate that unlike with the simple hybrids (Gly and Pro), when alkaline phosphatase is inserted as a sandwich near the short hydrophilic loop, it is not fully translocated to the periplasmic space. One possible explanation for the low activity is that the alkaline phosphatase insert destroys the functional assembly of Mdr, because the loop containing the insert is too short and may not be able to promote correct organization with such a long polypeptide insert. If this is the case, it is anticipated that the transport activity mediated by Mdr containing such an insert will be drastically affected. Accordingly, we compared tetraphenylarsonium (TPA) transport in cells expressing the sandwich hybrids SanV717 and SanL226 (Bibi and Béj, 1994), to cells expressing full-length Mdr-alkaline phosphatase hybrid (Ser) or native Mdr. Mdr-mediated TPA transport causes a lower level of accumulation of the substrate (Bibi et al., 1993). In contrast to SanL226, which exhibits wild-type activity as judged by the low level of TPA accumulation, SanV717 is completely inactive (Fig. 3). The low transport activity mediated by sandwich hybrid SanV717 is not due to low level of expression as demonstrated by a semiquantitative immunoblot analysis were the expression level of SanV717 was found similar to that of native Mdr, but much higher than SanL226 (data not shown).


Figure 3: Transport of TPA by E. coli UT5600 harboring pT7-5, pTSanL226, pTSanV717, pTS1276, or pTmdr1 (Bibi et al., 1993). Cells were grown, induced with isopropyl-1-thio--D-galactopyranoside, and treated with EDTA as described in Bibi et al. (1993). Transport of [H]TPA (23.8 mCi/mmol) at a final concentration of 0.4 mM was assayed at 34 °C by rapid filtration.



The notion that TM7 is actually two transmembrane segments suggests a major change in the orientation of the following transmembrane segment (indicated by an arrow in Fig. 2). To examine this possibility, we constructed hybrids in which alkaline phosphatase is fused to Mdr at residues Ala and Tyr in a region following the next hydrophobic segment (Fig. 5). As shown in Fig. 1C, hybrids Ala and Tyr exhibit high levels of normalized alkaline phosphatase activities, clearly suggesting that the next transmembrane segment flanked by amino acid residues Cys and Ala has an opposite orientation from the predicted structure and further supporting the proposal that the original TM7 is in fact composed of two transmembrane segments. These results are consistent with those obtained with a Mdr-prolactin hybrid in which prolactin is connected to amino acid residue 816 following TM8 (Skach et al., 1993). In that construct, prolactin is detected in the lumen of the microsomal membranes, in agreement with the results presented here and with the notion that TM8 crosses the membrane but in an opposite orientation.

Although topological data for most members of the ABC superfamily are not available, detailed studies have been carried out with the maltose periplasmic permease (see, for example, Prinz and Beckwith(1994); Boyd et al., 1993). The maltose transport system of E. coli is composed of two integral membrane proteins (MalG and MalF), a peripheral ATP binding subunit (MalK), and a periplasmic component (maltose-binding protein). Although the membrane components of the maltose transport system do not exhibit significant homology at the amino acid level with the membrane domains of Mdr, a striking similarity in their hydropathy profiles is apparent (Fig. 4). The greatest similarity exists between the hydropathy profiles of the N terminus of MalG and the N-terminal membrane domain of Mdr. In addition, the hydropathy profile of the N-terminal portion of MalF is similar to the respective region in the C-terminal membrane domain of Mdr (Fig. 4). Extensive study of MalF topology, reveals three TMs in this region of the protein (Boyd et al., 1993). Importantly, the results presented here indicate that the analogous portion of Mdr also contains three TMs (Fig. 5). The similar organization in both systems may be attributed to the role of the N-terminal sequences in membrane targeting and insertion. Such a role for the N-terminal sequence of the C-terminal membrane domain of Mdr was recently suggested when mutations in this region were shown to abolish proper processing and targeting (Loo and Clarke, 1994).


Figure 4: Comparison between the hydropathy profiles of the membrane components of the maltose transport system and Mdr. See the legend to Fig. 2 for details.



Although our proposal that TM7 consists of two TMs is supported by results presented both here and with other experimental systems (Skach et al., 1993; Zhang and Ling, 1991), it is inconsistent with epitope mapping studies using monoclonal antibodies (mAb) MRK-16 (Georges et al., 1993) and MM4.17 (Cianfriglia et al., 1994). Using these mAbs, it was proposed that a stretch of amino acid residues 740-747 is extracellular. An explanation for the discrepancy is not readily obvious, but may be provided in a recent report by Jachez et al.(1994). In their study they demonstrate that the extracellular binding levels of the MM4.17/MRK-16 epitope with the mAbs is temperature-dependent (4-fold higher at 37 °C than at 4 °C), unlike the binding properties of another cell surface epitope that is temperature-independent (MC57). In this regard, we speculate that unlike the interaction with antibodies, gene fusion experiments provide topological information at the time it is created cotranslationally, during the biosynthesis of the protein and its insertion into the membrane. Thus, although we propose a new topology, it is possible that this specific region of Mdr is subjected to reversible conformational changes in vivo, imposed by substrates or other unknown effectors as recently suggested for the effect of voltage on the open state conformation of a voltage-dependent channel (Slatin et al., 1994). Mobility of membranous protein segments is thought to involve unfavored energetic pathways. However, assuming that these energetic problems could be accommodated for by intra- or intermolecular interactions, we cannot rule out ``conformational adaptability'' (Theorell, 1967) in complex membrane translocators.


FOOTNOTES

*
This research was supported by the MINERVA Foundation, Munich/Germany and by a research grant from the Ebner family Biomedical Research Foundation at the Weizmann Institute of Science in memory of Alfred Ebner. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Incumbent of Dr. Samuel O. Freedman Career Development Chair in the Life Sciences. To whom correspondence should be addressed. Fax: 972-8-344118.

The abbreviations used are: Mdr, multidrug resistance protein; TM, transmembrane helix; mAb, monoclonal antibody; TPA, tetraphenylarsonium.


ACKNOWLEDGEMENTS

We are grateful to Dr. H. Ronald Kaback and Dr. Steve Karlish for helpful discussions during this work and to Dr. H. Ronald Kaback for his critical reading of the manuscript. We thank Dr. Ophry Pines for his valuable advice during the preparation of this manuscript and Hadassa Waterman for her assistance during the rotation studies.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.