©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Dissection of P-glycoprotein Nucleotide-binding Domains in Chimeric and Mutant Proteins
MODULATION OF DRUG RESISTANCE PROFILES (*)

(Received for publication, April 5, 1995)

Lucille Beaudet (§) Philippe Gros (¶)

From the Department of Biochemistry, McGill University, 3655 Drummond St., Montréal, Québec H3G 1Y6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We wished to determine if the two nucleotide-binding domains (NBD) of P-glycoprotein are functionally equivalent and interchangeable, and if not, which segments and amino acids are important for proper function of each NBD within the context of the C- or N-terminal P-glycoprotein halves. For this, we constructed and tested the biological activity in yeast and mammalian cells of a series of chimeric mdr3 cDNAs in which discrete domains of the N-terminal NBD (NBD1) were replaced by the homologous segments of the C-terminal NBD (NBD2). Although most NBD1 segments could be replaced without loss of P-glycoprotein function, exchange of small segments near the Walker B motif caused a dramatic reduction in Adriamycin, actinomycin D, and colchicine resistance in LR73 cells, as well as in FK506 resistance and STE6 complementation in yeast. Site-directed mutagenesis identified amino acid positions 522-525 (ERGA DKGT) and 578 (Thr Cys) as essential for proper function of NBD1 in the context of the N-terminal half P-glycoprotein. In addition, the observed phenotype of the mutants (altered drug resistance profile) suggests that these residues may participate directly or indirectly in substrate interactions and are possibly implicated in signal transduction from NBDs to transmembrane domains, the primary sites of drug binding in P-glycoprotein.


INTRODUCTION

Multidrug resistance in cultured cells in vitro and malignant tumors in vivo is caused by the expression of a membrane phosphoglycoprotein, called P-glycoprotein (reviewed in Refs. 1 and 2). P-glycoproteins are encoded by a small family of two genes in human (MDR1, MDR2), and three genes in mouse (mdr1, mdr2, mdr3). Transfection experiments have distinguished two classes of mdr genes: those directly conveying drug resistance (mouse mdr1/mdr3, human MDR1) and those unable to do so (mouse mdr2, human MDR2) (reviewed in (3) ). P-glycoprotein binds photoactivatable drug (4) and ATP (5, 6) analogs, causes a reduction in intracellular drug accumulation, and has been proposed to function as a drug transporter(7) . Amino acid sequence analysis and hydropathy profiling indicate that P-glycoprotein is formed by two homologous halves, each composed of 6 transmembrane (TM)()domains and one nucleotide-binding domain (NBD)(8, 9) . This structural unit has been evolutionarly conserved in a large number of prokaryotic and eukaryotic membrane proteins known as the ABC (ATP-Binding Cassette) superfamily of transporters(10) . In prokaryotes, this group of transporters can import or export, at the expense of ATP, a large variety of structurally unrelated substrates such as sugars, amino acids, peptides, ions, and others(11) . In eukaryotes, the best characterized ABC transporters include the yeast ``a'' mating pheromone transporter Ste6(12) , the pfmdr1 protein associated with chloroquine resistance in the malarial parasite Plasmodium falciparum(13) , the CFTR chloride channel, the mutation of which causes cystic fibrosis in human(14) , and the TAP1 and TAP2 peptide pumps participating in antigen presentation to cytotoxic T lymphocytes (15, 16, 17, 18) . The structural homology detected between ABC transporters also translates into functional similarity since mdr3 can partially complement STE6, thus restoring mating in a ste6 sterile yeast strain(19) .

The exact mechanism by which P-glycoprotein mediates reduction of drug accumulation remains controversial. Although several studies in homologous and heterologous systems have demonstrated active drug transport by P-glycoprotein(2, 7, 20) , other observations have suggested rather indirect or secondary mechanisms of drug transport for P-glycoprotein (reviewed in (21) ). In addition, the recent demonstration that P-glycoprotein encoded by mdr2 functions as a lipid translocase (22) suggests a potential flippase mechanism for mdr1- and mdr3-mediated drug transport(22, 23) .

In the absence of three-dimensional structural information, the identification of structure/function relationships in P-glycoprotein has relied on the scrutiny of P-glycoprotein primary sequence and on biochemical and genetic analysis. Although the exact topology and number of TM segments of P-glycoprotein remain to be fully defined, considerable evidence indicates that the hydrophobic domains are intimately associated with drug binding. First, energy transfer experiments using membrane probes and MDR drugs indicate that drug molecules interact with P-glycoprotein within the cell membrane(24) . Similar conclusions were drawn from a study in which extrusion of fluorescent cellular indicators from drug-resistant cells was monitored (25) and from experiments where forskolin-binding to P-glycoprotein was analyzed(26) . Also, epitope mapping studies of proteolytic P-glycoprotein fragments labeled with photoactivatable drug analogs identify two small 4-5-kDa peptides as drug-binding sites located within or immediately C-terminal to TM6 and TM12(27) . Finally, analysis of P-glycoprotein mutants showing alterations in drug resistance profiles reveals that mutations near or within TM domains affect substrate specificity(28, 29, 30, 31, 32, 33, 34) . The key role of TM domains in substrate recognition and binding has also been established for CFTR and TAP1/TAP2, where mutations near or within these segments affect ion selectivity of the channel (35) and peptide specificity of the pump (36) , respectively.

The two nucleotide-binding sites in P-glycoprotein are defined by the presence of consensus Walker A (GXGKS/T) and Walker B (R/KXLHydD, where Hyd is a hydrophobic residue), motifs previously described for many nucleotide-binding proteins and ATPases(37) . The lysine present in the Walker A motif is believed to interact with the phosphate group of ATP while the B motif might form a hydrophobic pocket housing the adenine moiety of the nucleotide(38) . In addition, ABC transporters present a third consensus sequence, the linker peptide, preceding the B motif and defined by the sequence LSGGXRHydXHydA(39) . Computer modelling studies based on the structure of adenylate kinase predict the presence of additional structural features specific to ABC transporters(10, 40) . These ``helical region''(40) , and ``loops 2 and 3''(10) , are located between the A motif and linker peptide and have been proposed to play a role in energy-coupling from the NBDs to the TM regions to effect transport(10, 40) .

P-glycoprotein-mediated drug transport requires the presence of cellular ATP(41, 42) , and a drug-stimulatable ATPase activity for P-glycoprotein has been characterized by several groups ( (43) and references within). Analysis of half-protein expression demonstrates that each of the two NBDs of P-glycoprotein can hydrolyze ATP(44) . Studies of chimeras constructed between mdr1 and mdr2 indicate that corresponding NBDs from the two P-glycoprotein isoforms are exchangeable (Mdr2 NDB1 Mdr1 NBD1; Mdr2 NBD2 Mdr1 NBD2) and are an important component of the common mechanism of action of the different P-glycoprotein isoforms(45) . In addition, mutations introduced in the A motif of either of the two P-glycoprotein NBDs completely abrogate drug resistance, stressing the key role of these sequences in P-glycoprotein function(46) . Also supporting the functional importance of each NBD is the observation that either naturally occurring, or experimentally introduced mutations in either NBD of other ABC transporters such as CFTR (47) and Ste6 (48) abolish or greatly reduce their activity.

One of the unresolved issues concerning the function of P-glycoprotein is whether both NBDs are equivalent, and thus interchangeable, or whether each functions only within the context of a specific membrane environment provided by TM domains. Identifying the intramolecular interactions between the NBDs and their environment may help elucidate the mechanism of energy transfer from the NBDs to the TM regions to mediate transport. A considerable body of data suggests that cooperative interactions involving both sets of TM domains and NBDs of P-glycoprotein (and of other ABC transporters) are required for transport. For example, photolabeling studies with drug analogs identify drug-binding sites in both TM regions of P-glycoprotein(27) , and mutations in either of the two Walker A motifs cause a complete loss rather than a reduction of function(46) . Likewise, independent expression of either half of Ste6 fails to create a functional transporter, while expression of both polypeptides in the same cell restores a-factor transport and mating(48) . Many experiments further indicate that functional differences exist between the two NBDs of ABC transporters. In Ste6, mutations in NBD1 have a more deleterious effect on function than mutations at the homologous residue of NBD2(48) . In CFTR, expression of the N-, but not of the C-terminal half of the protein, results in the appearance of a fully regulated channel(49) . Also, competitive inhibition of CFTR function by ADP seems to occur mostly through interactions with NBD2 but not NBD1(50) , and equivalent mutations in NBD1 and NBD2 of CFTR produce opposite effects on sensitivity of the channel to activation by forskolin(51) .

The goals of the present study were first to determine whether both NBDs of P-glycoprotein are functionally equivalent, and then to identify the protein segments and amino acids that are not interchangeable and are therefore likely to be involved in specific interactions with the environment in which each NBD is fully active.


MATERIALS AND METHODS

Construction of Chimeric and Mutant mdr3 cDNAs

Plasmid pDR16 (52) in which the full-length mdr3 cDNA is inserted into the SmaI site of the pGEM7Zf vector (Promega, Canada) was used as template in this study. To facilitate the construction of mutations in NBD1 or chimeric molecules in which parts of NBD1 were replaced with the corresponding segment from NBD2, two unique restriction sites flanking NBD1 were inserted by site-directed mutagenesis. For this, a 2.2-kilobase EcoRI mdr3 fragment (5`-half: polylinker to position 2249) was subcloned into phage vector M13 mp18, and silent mutations creating NruI (position 1346) and SalI sites (position 1908) were introduced by site-directed mutagenesis (Table 2), using a commercially available kit (Amersham, Canada). The integrity of the BalI to EcoRI mdr3 fragment (positions 798-2249) containing the novel NruI/SalI restriction cassette was verified by sequencing prior to recloning into pDR16. The modified mdr3 cDNA was excised from pDR16 by digesting with SphI and ClaI, and its extremities were repaired with T4 DNA polymerase. It was then inserted into the mammalian expression vector pEMC2b (generous gift of Dr. R. Kaufman, Yale University, New Haven, CT) (pEMC-mdr3), and into the yeast expression vector pVT101-U (pVT; (53) ) from which the NruI site had been destroyed (pVT-mdr3).



The general strategy for the construction of chimeric mdr3 cDNAs relied on the use of hybrid oligonucleotide primers and PCR amplification (54) to fuse non-contiguous DNA segments (Fragments 1, 2, 3; Table 1) in two rounds of PCR. In all chimeric constructs, a NBD1 fragment was replaced by the homologous segment of NBD2, while NBD2 was left intact. The strategy and protocol were as follows. First, individual NBD2 fragments to be duplicated (Fragments 2) were amplified by PCR using an mdr3 3` half-template and complementary oligonucleotide primers described in Table 1(e.g. chimera A, primers 3326s and 3409a). Similarly, fragments from NBD1 flanking individual Fragments 2 on either side were PCR amplified from an mdr3 5` half-template. The upstream 5` fragments (Fragments 1) were generated using one primer located 5` of the NruI site (chimera A, primer 1295s) and an antisense chimeric primer in which 12 nucleotides at the 5` end were complementary to the corresponding Fragment 2 and 12 nucleotides at the 3` end complementary to the adjacent NBD1 sequence (chimera A, primer 1402a). Likewise, the downstream 3` fragments (Fragments 3) were generated using one antisense primer located downstream of the SalI site (chimera A, primer 2027a), and a sense chimeric primer in which 12 nucleotides at the 5` end were complementary to individual Fragments 2 and the 12 at the 5` end complementary to the adjacent NBD1 sequence (chimera A, primer 1481s). To construct individual mdr3 cDNAs with chimeric NBD1 segments, the NBD2-derived Fragment 2 was mixed separately with either adjacent Fragments 1 or 3 in a buffer consisting of 7 mM Tris (pH 7.5), 50 mM NaCl, and 7 mM MgCl. The DNA fragments were heat denatured (95 °C, 5 min), and complementary ends were allowed to re-anneal (37 °C for 15 min) before being filled-in using the Klenow fragment of DNA polymerase I and dNTPs (50 µM). Double-stranded hybrid molecules were recovered by phenol-chloroform extraction and ethanol precipitation, and the 1-2 and 2-3 chimeric molecules were PCR amplified using the proximal primer from Fragment 1 plus the 1-2 hybrid primer (chimera A, primers 1295s and 3409a) or the 2-3 hybrid primer plus the distal primer from Fragment 3 (chimera A, primers 3326s and 2027a), respectively. Finally, the 1-2-3 chimeric molecule was created by a similar strategy using gel purified 1-2 and 2-3 hybrid fragments mixed and amplified with the proximal primer from Fragment 1 and the distal primer from Fragment 3 (chimera A, primers 1295s and 2027a). Fragment 1-2-3 was then digested with NruI and SalI, gel-purified, and cloned into the corresponding cassette of pVT-mdr3 or pEMC-mdr3. All amplification reactions were carried out for 20 cycles (1 min at 94 °C, 1 min at 45 °C, and 1 min at 72 °C) in a buffer consisting of 20 mM Tris (pH 8), bovine serum albumin (1 mg/ml), 50 mM NaCl, 200 µM of each dNTP, MgCl at concentrations ranging from 0.5 to 1.5 mM, 0.5 units of Vent polymerase (New England Biolabs, Canada), and 0.5 ng of template. The integrity of the nucleotide sequence of PCR-amplified fragments and of restriction enzyme sites used to produce all constructs were ascertained.



mdr3 mutants bearing discrete substitutions in NBD1 were generated by site-directed mutagenesis using a commercially available system (Amersham, Canada). For this, single-stranded DNA template from the 2.2-kilobase EcoRI mdr3 fragment served as a template for creating mutants R488D/E489N/D490S/RV, N544Q, K546H, N544Q/K546H, E522D/R523K/A525T, A563K, T578C, and A567E. Mutant T578C was used as a DNA template for creating mutants E522D/R523K/A525T/T578C. All oligonucleotides used for mutagenesis are listed in Table 2.

Tissue Culture, Transfections, and Drug Cytotoxicity Assays

All cell populations were maintained in -minimal essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (50 units/ml) and streptomycin (50 µg/ml) (complete medium). Wild-type (WT), chimeric, and mutant mdr3 cDNAs cloned in pEMC2b were introduced into Chinese hamster ovary cells LR73 by co-transfection with marker plasmid pSV2neo(55) , as described previously(46) . Co-transfectants were selected for 10 days in medium containing Geneticin (G418; Life Technologies, Inc./BRL, Canada) at a final concentration of 500 µg/ml, harvested as mass populations, and stable mdr3 transfectants were further selected in complete medium containing vinblastine (VBL, 25 ng/ml) (Sigma). VBL resistant (VBL) cells were harvested as mass populations, expanded, and frozen as several aliquots which were thawed out before each experiment.

Drug cytotoxicity assays were carried out using a method based on cell protein staining by sulforhodamine B, as described previously(56) . In these experiments, a G418 mass population of cells co-transfected with an mdr3 cDNA cloned in pEMC2b in the antisense orientation were used as a negative control. Briefly, 5 10 cells from each mass population were plated in duplicate in 96-well titer plates containing increasing concentrations of either VBL, colchicine (COL) (Sigma), Adriamycin (Royal-Victoria Hospital, Montréal, Canada), or actinomycin D (ACT) (Royal-Victoria Hospital, Montréal, Canada). After a 3-day incubation at 37 °C, cells were fixed in 17% trichloroacetic acid, and stained with a solution of 0.4% sulforhodamine B in 1% acetic acid. Excess stain was removed by five washes in 1% acetic acid and plates were air-dried overnight. The stain was dissolved in 200 µl of Tris (10 mM, pH 9) and quantitated with an enzyme-linked immunosorbent assay plate reader (Bio-Rad, model 450) set at 490 nm. The relative plating efficiency represents the ratio of the absorbance measured at a given drug concentration divided by the absorbance of the same population of cells plated in the absence of drug. The D is defined as the drug concentration required to reduce the relative plating efficiency of each cell population by 50%.

Testing of mdr3 Function in Yeast Cells

The biological activity of pVT-mdr3 constructs was also tested by their capacity to restore mating in a ste6 sterile yeast strain, and to confer cellular resistance to the fungicidal compound FK506. Both assays were done in Saccharomyces cerevisiae strain JPY201 (MATaste6ura3) which has been described previously(57) . Strain DC17 (MAThis1) was used as the tester strain in the mating assays. Strains JPY201 and DC17 were kindly provided by Dr. M. Raymond (Institut de Recherches Cliniques de Montréal, Montréal, Canada). JPY201 cells were transformed with the various pVT-mdr3 constructs by the lithium-acetate method(58) , and pools of ura colonies were harvested, expanded in culture, and kept frozen until used in each experiment. YPD-rich medium, minimal medium, and synthetic medium lacking uracil (S.D.-ura) were prepared as described(59) . The mating efficiency of pVT-mdr3 yeast transformants was quantified by filter assay(60) , according to a procedure we have described previously(61) . Mating efficiency was expressed as the ratio of diploid colonies grown on minimal plates to the number of haploid JPY201 transformants introduced in the mating reaction, and is also expressed as the percentage of the mating efficiency of WT mdr3 transformants.

Cellular resistance of yeast transformants expressing either WT or mutant mdr3 cDNAs was estimated by a growth inhibition assay using the antifungal peptide macrolite FK506, previously shown to be a P-glycoprotein substrate in yeast(61) . Stock solutions of FK506 (10 mg/ml) were prepared in methanol and kept at -80 °C until use. The growth inhibition assay was carried out essentially as described(61) . Briefly, fresh overnight cultures of pVT-mdr3 transformants grown in S.D.-ura were diluted to optical density 0.005 (600 nm) in YPD medium, and added (50 µl) to an equal volume of YPD medium containing 100 µg/ml FK506 (final concentration of 50 µg/ml) in a 96-well microwell titer plate. The plates were wrapped with Parafilm to prevent evaporation and incubated at 30 °C with constant agitation (250 rpm). Growth was monitored by optical density measurement using an enzyme-linked immunosorbent assay plate reader (Bio-Rad, model 450) set at 490 nm. The growth rate of the various mdr3 transformants was calculated in the exponential growth phase over a 4.5-h period. Growth inhibition by FK506 was measured as a reduction in growth rate which was expressed as a percentage of control transformants expressing WT mdr3.

Immunoblotting and Immunoprecipitation

Membrane-enriched fractions were prepared from mass populations of VBL LR73 transfectants expressing individual WT or mutant mdr3 cDNAs, as described previously(52) . Protein concentrations were determined using a commercially available reagent (Bio-Rad) as described(52) . Crude membrane proteins (6.5 µg) were loaded onto a 7.5% polyacrylamide gel containing SDS (SDS-polyacrylamide gel electrophoresis) and transferred onto a nitrocellulose membrane by electroblotting. The resulting blots were blocked (16 h at 4 °C) with a solution containing 1% bovine serum albumin (Fraction V), 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20, and incubated (1 h at 25 °C) with the mouse anti-P-glycoprotein monoclonal antibody C-219 (Centocor Corp., Philadelphia, PA), used at a 1:300 dilution. Specific immune complexes were detected using a second goat anti-mouse antibody coupled to alkaline phosphatase (Jackson ImmunoResearch Inc.) used at a 1/3500 dilution, and revealed with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine and nitro blue tetrazolium substrates according to the manufacturer's recommendations (Life Technologies, Inc./BRL, Canada).

Immunoprecipitation of P-glycoproteins encoded by WT and mutant mdr3 cDNAs from yeast transformants was carried out as described elsewhere(61) , using the isoform-specific rabbit anti-Mdr3 polyclonal antibody 2037 (62) at a 1:100 dilution. Yeast cells were labeled for 1 h with 100 µCi of [S]methionine (1175 Ci/mmol; Du Pont Canada). Immune complexes were purified using Protein A-Sepharose beads (Pharmacia Canada), dissolved in Laemmli buffer(63) , and incubated at 25 °C for 30 min prior to SDS-polyacrylamide gel elcetrophoresis. After electrophoresis, the gel was fixed (30% methanol, 10% acetic acid), and treated with a fluorography enhancer (Amplify; Amersham Canada) as recommended by the manufacturer. The gel was dried and exposed to Kodak X-AR film for 72 h at -80 °C.


RESULTS

Sequence Comparison of the Two Nucleotide-binding Domains of Mouse Mdr3

Amino acid alignment of the two mouse Mdr3 NBDs (NBD1, positions 419-579; NBD2, positions 1062-1224) indicates that they share 66% identity with an additional 15% conservative substitutions for an overall similarity of 81% (Fig. 1). Both sets of Walker A and B motifs conform to the consensus sequences(37) . Only a single N (position 424) to S (position 1067) conservative substitution within the A motif, and two non-conservative substitutions in the B motif (N544/Q1189 and K546/H1191) distinguish NBD1 from NBD2 in these regions. The linker peptides are identical in both NBDs (LSGGQKQRIAIA). Likewise, the 28-amino acid residue stretch located immediately C-terminal to the B motif (positions 552/1197 to 579/1224) is highly conserved in both NBDs (25/28 identical residues). In contrast, the N- and C-terminal central domains, lying between the A motifs and the linker peptides (positions 431/1074 to 526/1171), share a lower degree of sequence identity (51% identity, 23% similarity), and contain several short clusters of non-conservative substitutions within the second half of the central region (positions 477/1119 to 519/1156). Notably, this section contains two extra residues at positions 1134-1135 (R-V), compared to the sequence RED found at positions 488-490. Taken together, these observations indicate that the regions of homology and divergence between the two NBD sites of P-glycoprotein are not randomly distributed but rather are restricted to specific subregions of these domains.


Figure 1: Amino acid sequence alignment of the two mouse Mdr3 nucleotide-binding domains. Amino acid numbering is according to (15) . Walker A and B motifs are indicated, as well as the position of the NBD1 segments replaced by their NBD2 counterpart in the individual chimeras. Solid line, identical residues between the two NBDs; two dots, conserved substitutions.



Construction of Chimeric mdr3 cDNAs with Modifications in NBD1

We wished to determine whether the two NBDs of P-glycoprotein were structurally/functionally equivalent, and thus interchangeable, or if they could only function within a distinct sequence context, perhaps provided by each P-glycoprotein half. We generated a set of seven chimeric and one mutant mdr3 cDNAs in which discrete portions of NBD1 had been replaced by the homologous segments of NBD2 (Fig. 2). In chimera F, the entire NBD1 (including the A and B motifs plus the central region; positions 419-579) was replaced by the corresponding segment of NBD2 (positions 1062-1224). Walker A (GXGKS/T) and Walker B (R/KXLHydD) motifs were also exchanged in independent chimeras. In chimera A, the NBD1 A motif and a few surrounding residues (positions 419-443) were replaced by the corresponding residues of NBD2 (7/25 non-identical residues). In chimera D, the B motif, linker peptide, and a short C-terminal portion of the central region (positions 522-562) were exchanged for NBD2 homologous sequences (5/41 non-identical residues). To analyze the central region, chimeras B and C, and mutant R488D/E489N/D490S/RV were generated. Chimera B contains, in NBD1, the NBD2 A motif followed by approximately half of the NBD2 central region (positions 419-476; 23/58 substitutions). In chimera C, the rest of the NBD2 central region, up to the boundary of chimera D, is inserted into NBD1 (positions 477-521), and includes the majority of the non-conservative substitutions (25/45 non-identical residues, 19/45 non-conservative substitutions). In mutant R488D/E489N/D490S/RV, the short segment of non-identity in the two NBDs located from positions 488/1131 to 490/1135 was analyzed. Finally, the highly conserved region located immediately C-terminal to the B motif was studied in chimeras E and G. Chimera E contains the segment exchanged in chimera D plus an additional 17 C-terminal residues (positions 522-579; 8/58 non-identical residues), while in chimera G only the latter segment was replaced (positions 563-579; 3/17 non-identical residues).


Figure 2: Schematic representation of chimeric and mutant Mdr3 proteins. A, schematic representation of structural domains in the Mdr3 protein includes transmembrane segments (TM1-6, 7- 12), nucleotide-binding domains (NBD1, NBD2), and linker region (L). Chimeric constructs (A to G) in which NBD1 segments were replaced by the corresponding NBD2 segments (hatched boxes) are shown. Enlarged section represents the entire NBD1 from the end of TM domain 6 to the beginning of the linker region. Chimeras A to F were generated using a three-fragment PCR-amplification technique described under ``Materials and Methods,'' while mutant RED DNSRV was created by site-directed mutagenesis. Positions of the Walker A and B motifs are identified by black boxes. B, identification of non-conserved residues between NBD1 and NBD2 within the segments exchanged in chimeras D, E, and G. Individual and double mutants were created by site-directed mutagenesis at indicated positions.



Drug Resistance Profiles of LR73 Cells Expressing Chimeric mdr3 cDNAs

WT, chimeric, and R488D/E489N/D490S/RV mutant mdr3 cDNAs cloned into the expression vector pEMC2b were introduced into LR73 cells by co-transfection with the indicator plasmid pSV2neo. Mass populations of G418 resistant (G418) clones co-transfected with individual mdr3 constructs were harvested and plated in medium containing vinblastine (VBL; 25 ng/ml) to evaluate their potential to convey drug resistance. Cells co-transfected with chimera F failed to give rise to VBL resistant (VBL) colonies, indicating that transfer of the complete NBD2 segment into NBD1 results in a non-functional protein. In contrast, mass populations of cells co-transfected with the other chimeric and mutant cDNAs all gave rise to VBL colonies, indicating that the exchange of small segments within the first NBD does not affect the transport capacity of the resulting chimeric P-glycoprotein. The VBL phenotype observed for all chimeric and mutant mdr3 constructs (except chimera F) also suggested that the corresponding proteins were properly targeted to the plasma membrane. This proposition was supported by immunoblotting experiments. Results in Fig. 3A (lanes 1-8) revealed the presence of an immunoreactive P-glycoprotein species of approximately 160 kDa in enriched membrane fractions from all mass populations which was absent from control LR73 cells transfected with the mdr3 cDNA cloned in the reverse orientation (lane 17). Although not identical, the amount of Mdr3 expressed in the various transfectants was similar. Taken together, these results indicate that replacement of small segments of NBD1 by the corresponding segments of NBD2 does not alter the transport capacity of the resulting proteins and does not seem to have major effects on protein targeting to the plasma membrane.


Figure 3: Immunodetection of WT, chimeric, or mutant Mdr3 proteins in mammalian and yeast cells. A, Western blot analysis of membrane-enriched preparations from VBL mass populations of co-transfected LR73 cells. The mouse anti-P-glycoprotein monoclonal antibody C219 was used to detect Mdr3 protein expression. A G418 mass population of cells co-transfected with WT mdr3 cDNA cloned in the reverse orientation into vector pEMC2b was used as negative control. B, immunoprecipitation of Mdr3 from yeast transformants metabolically labeled with [S]methionine using the polyclonal anti-Mdr3 isoform-specific antiserum 2037(62) .



We tested the possibility that replacements in NBD1 may have more subtle effects on Mdr3 function, perhaps affecting the spectrum of drug resistance conveyed by this protein. For this purpose, we assayed the various mass populations of VBL cells for their resistance to other MDR drugs, namely colchicine (COL), Adriamycin, and actinomycin D (ACT). Cytotoxicity levels were expressed as D. The relative resistance was expressed as fold resistance over background and was calculated by dividing the D of transfectants over the D of control cells. Results are summarized in Fig. 4and Table 3. In agreement with our preliminary observations, cytotoxicity assays revealed that all chimeric mdr3 cDNAs, including mutant R488D/E489N/D490S/RV, conferred similar levels of vinblastine resistance (22-30-fold over background). However, detailed analysis of transfectant resistance profiles to the other MDR drugs revealed dramatic differences. Chimera B and mutant R488D/E489N/D490S/RV were the only constructs with drug resistance profiles similar to WT mdr3. All other chimeras showed significant deviations from the WT profile. Chimera A transfectants displayed a modest but significant increase in resistance to Adriamycin (31 versus 15 ), COL (31 versus 18 ), and ACT (53 versus 33 ), while chimera C transfectants showed similar increased resistance to Adriamycin (31 versus 15 ) and COL (36 versus 18 ), but not ACT. Even more striking deviations from the WT profile were noted for transfectants expressing chimeras D, E, and G. Although these transfectants produced VBL resistance levels similar to WT mdr3, their levels of resistance to Adriamycin, COL, and ACT were significantly reduced. In particular, chimera E transfectants showed very low levels resistance to COL (3 ) and ACT (5 ), and resistance to Adriamycin in these cells was barely over background (2 ). Likewise, chimera G transfectants displayed only low levels of resistance to COL and Adriamycin (4 ), while chimera D transfectants showed, compared to WT mdr3, a 2-fold decrease in resistance to Adriamycin, COL, and ACT. These results suggest that the replacement of short segments of NBD1 overlapping the Walker B motif with the corresponding segments from NBD2 has strong effects on drug specificity but does not affect the transport capacity of Mdr3 since VBL resistance is retained.


Figure 4: Multidrug resistance profiles of LR73 cells transfected with WT, chimeric, or mutant mdr3 cDNAs. D values for drugs VBL (panel A), COL (panel B), Adriamycin (panel C), and ACT (panel D) were determined as described in Table 3and are presented with the corresponding standard deviations. Shaded bars, transfectants with drug resistance profiles close to WT; hatched bars, transfectants with increased resistance to one or more drugs; black bars, transfectants with decreased resistance to COL, Adriamycin, and ACT. Chimera F transfectants did not survive VBL drug selection and could not be analyzed for the three other drugs. The corresponding chimeric and mutant mdr3 cDNAs tested are represented schematically (see legend to Fig. 2for details).





These cytotoxicity assays were performed on cell populations that had been preselected in low level VBL (25 ng/ml) in order to eliminate all the non-expressing G418 cells and thus allow quantitative determination of plating efficiency. To determine if VBL preselection of the cells did bias the unique pattern of drug resistance detected in certain chimeras, cells from the G418 mass populations co-transfected with WT mdr3 or chimeras C, D, E, G, or F were plated in medium containing VBL (25 and 100 ng/ml), COL (100 and 500 ng/ml), ACT (10 and 50 ng/ml), or Adriamycin (50 and 250 ng/ml), and the fraction of surviving cells was determined seven days (Fig. 5, left panel) or 10 days (Fig. 5, right panel) later. This experiment clearly showed that mass populations of chimera F transfectants display no resistance to COL, Adriamycin, or ACT and that chimera D, G, and E transfectants, although expressing levels of VBL resistance similar to WT, display a dramatic reduction in COL, ACT, and Adriamycin resistance as compared to WT transfectants. The concordance of drug resistance profiles observed among the various chimeras in G418 mass populations and in cells preselected in VBL-containing medium, clearly establishes that VBL preselection did not influence the drug resistance profiles displayed by the chimeras.


Figure 5: Drug resistance profiles of G418 mass populations of transfectants. G418 mass populations of cells co-transfected with either WT mdr3 or chimeras C, D, G, E, and F were seeded (2.5 10 cells) in medium containing VBL (25 and 100 ng/ml), COL (100 and 500 ng/ml), Adriamycin (50 and 250 ng/ml), or ACT (10 and 50 ng/ml). 2.5 10 cells were seeded in control wells containing drug-free medium. Cells were stained with crystal violet (1% w/v in 20% ethanol) 7 days (no drug and lower concentrations) or 10 days (higher concentrations) later. G418 cells co-transfected with the mdr3 cDNA cloned in the antisense orientation were used as negative controls.



Drug Resistance Profiles of LR73 Cells Expressing Mutant mdr3 cDNAs

The 58-residue segment of NBD1 (positions 522-579) replaced in chimeras D, E, and G differs from its NBD2 counterpart by only 8 residues. We therefore generated a series of mutants to further identify the NBD2 residues that were influencing substrate specificity when put into NBD1. Mutants N544Q, K546H, N544Q/K546H, E522D/R523K/A525T, A563K, A567E, and T578C were created by site-directed mutagenesis (Fig. 2B), cloned into pEMC2b, and introduced into LR73 cells by co-transfection with pSV2neo. Each construct gave rise to VBL populations of cells, an observation in agreement with the VBL phenotype of the parental chimeras D, E, and G (Figs. 3 and 5). Immunoblotting experiments showed that similar amounts of mutant Mdr3 proteins were produced in the membrane fraction of these new transfectants (Fig. 3A, lanes 9-15). Cytotoxicity assays on the VBL mass populations demonstrated that individual mutations within the B motif (N544Q and K546H) had no significant effect on the drug resistance profile (Table 3). Combination of the two mutations (N544Q/K546H) slightly decreased resistance to COL, Adriamycin, and ACT, but to a lesser extent than chimera D (Table 3), indicating that the additional divergent residues in chimera D (E522D, R523K, and A525T) were contributing to the reduced resistance levels. Indeed, cytotoxicity assays revealed that cells expressing the corresponding mutant E522D/R523K/A525T display a resistance profile indistinguishable from that of chimera D, thus confirming the importance of this segment of four residues for the drug specificity of Mdr3.

To identify the specific residues responsible for the altered drug resistance displayed by chimera G transfectants, mutants A563K, A567E, and T578C were analyzed as described above. Determination of the drug resistance profile conveyed by these three individual mutants identified unambiguously the T578C mutation as responsible for the phenotype of chimera G (Table 3, Fig. 4). Indeed, cells expressing mutant T578C showed a drug resistance profile very similar to that observed for chimera G, while the mutant A563K and A567E multidrug resistance patterns were comparable to WT mdr3 (Table 3). Chimera E combines all the modifications present in chimera D and chimera G, and displays the lowest levels of COL, Adriamycin, and ACT resistance (Table 3, Fig. 4). To determine whether the E522D/R523K/A525T and T578C mutations are solely responsible for the altered drug resistance profile expressed by chimera E, the mutant E522D/R523K/A525T/T578C was created and analyzed (Table 3, Fig. 4). Results from drug cytotoxicity assays indicated that this was indeed the case since E522D/R523K/A525T/T578C and chimera E transfectants display identical drug resistance profiles. Taken together, these results identify the ERGA segment (positions 522-525) and Thr-578 as the only amino acids of NBD1 that cannot be replaced by their NBD2 counterparts without significant alteration of Mdr3 function. As for the chimeric constructs, substrate specificity rather than transport capacity (intact VBL resistance) is affected, suggesting that these residues may participate either directly or indirectly in the process of substrate recognition by Mdr3.

Expression and Characterization of Chimeric and Mutant Mouse mdr3 cDNAs in Yeast Cells

We wished to verify that the unique effect of mutations detected in chimeric and mutant Mdr3 after analysis in LR73 cells was not limited to this experimental system, but reflected a true modulation of Mdr3 activity. For this, we monitored the activity of Mdr3 in a yeast heterologous expression system. We have shown previously that mdr3 can complement the biological activity of the STE6 gene by restoring mating in a sterile yeast strain(19) , and that it can also confer, to yeast cells, resistance to the antifungal macrolites FK506 and FK520(61) . Chimeric and mutant mdr3 cDNAs were cloned into the expression vector pVT-U, transformed into S. cerevisiae strain JPY201, and mass populations of transformants were recovered from selective medium. Immunoprecipitation of [S]methionine-labeled cell lysates with the isoform-specific anti-mouse Mdr3 antiserum 2037 (62) showed that all yeast transformants expressed chimeric or mutant proteins (Fig. 3B).

Cellular resistance to the antifungal agent and P-glycoprotein substrate FK506 was evaluated for all transformants using a cell growth inhibition assay, as described previously(61) . Cell growth in the presence of FK506 was monitored (490 nm) for 22 h (Fig. 6A). The growth rate of each transformant population was calculated and expressed as the percentage of WT mdr3 growth rate (Fig. 6C). In drug free medium, all transformants showed identical growth profiles (data not shown) while in the presence of FK506, considerable variations in growth rate were observed (Fig. 6). Chimeras A and B showed growth rates that were identical to WT (Fig. 6C), and growth of chimera C transformants was near WT levels (Fig. 6C, 60% of WT mdr3). On the other hand, chimeras D and G displayed much weaker activity against FK506. Levels of resistance produced by chimeras F and E were the lowest, with the latter showing growth characteristics identical to that of the pVT negative control. Growth of the R488D/E489N/D490S/RV transformants was also impaired in FK506 with only 40% of the WT growth. Analysis of growth rates of NBD1 point mutants (Fig. 6, B and C) indicated that mutations at positions 544 and 546 either alone or in combination (N544Q, K546H, and N544Q/K546H), and mutations at positions 563 and 567 (A563K and A567E) had no major effect on FK506 resistance. However, cells expressing either the E522D/R523K/A525T or the T578C mutants grew significantly slower, and combination of both mutations in the same molecule abolished resistance to FK506 (Fig. 6, B and C). Interestingly, resistance of all the chimeric and mutant mdr3 transformants to FK506 appeared very similar to their respective resistance profiles for Adriamycin, COL, and ACT in LR73 cells (Fig. 4).


Figure 6: Growth of yeast transformants expressing WT, chimeric, or mutant mdr3 cDNAs in medium containing FK506. Yeast transformants were seeded in 96-well plates containing FK506 (50 µg/ml) as described under ``Materials and Methods.'' Growth, as measured by optical density (490 nm), was monitored over a 22-h period. A, growth curves of transformants expressing chimeras and mutant R488D/E489N/D490S/RV mdr3 cDNAs. B, growth curves of yeast transformants expressing mutant mdr3 cDNAs. C, growth rates were determined by measuring the slope of the growth curves over a 4.5-h period (between 16.5 and 21 h) and growth rate of WT mdr3 transformants was set at 100%. Growth rate of the yeast transformants expressing chimeric and mutant mdr3 cDNAs was calculated and expressed as a percentage of WT. The corresponding chimeric and mutant mdr3 cDNAs tested are represented schematically (see legend to Fig. 2for details).



We next measured the capacity of the various mdr3 chimeras and mutants to restore mating in the sterile ste6 yeast strain JPY201 (Table 4). Mating frequency was expressed as the fraction of transformed JPY201 cells introduced in the assay forming diploid colonies on selective medium. In this assay, Ste6 transformants have a mating frequency close to 1, indicating that every cell can fuse and become diploid. Percentage of WT Mdr3 mating frequency was calculated for all mutant and chimeras (Table 4). Chimera B was the only chimera with a mating efficiency comparable to that of WT (110%). Chimeras A and C were significantly more active with 680 and 1500% of WT activity, respectively. By contrast, the activity of the R488D/E489N/D490S/RV mutant was significantly reduced (less than 1% of WT) while chimeras D, E, F, and G were basically inactive in mating assays (more than 1000-fold less than WT frequency). Similar results were obtained with single colonies originating from an independant transformation (not shown), indicating that the observed variations were not due to the presence of contaminants in the pools of transfectants. NBD1 point mutants also showed a wide range of mating activities: some mutations had either no or rather small effects on mating (N544Q, A567E, N544Q/K546H) while others significantly decreased the mating activity compared to WT. The phenotype of A563K transformants was of interest since their mating efficiency was only 8% of WT, while their drug resistance phenotype had been previously observed to be as WT. More strikingly, mutants T578C and E522D/R523K/A525T, alone or in combination, showed no detectable mating activity, like chimeras D, E, F, and G. Taken together, our data indicate that the functional characteristics of chimeric and mutant Mdr3 proteins established in yeast cells correlate well with their drug resistance phenotype in LR73 cells, and suggest that, for Mdr3, the NBD1 residues ERGA and Thr-578 cannot be substituted for their NBD2 counterparts without loss of biological activity for certain substrates.




DISCUSSION

P-glycoprotein is composed of two symmetrical halves, each encoding six TM domains and one NBD, which share significant sequence homology and a common ancestral origin. In particular, the two predicted NBDs share over 80% sequence similarity. The functional significance of this structural arrangement in general, and the role of the two highly homologous NBDs, in particular, are unclear and were addressed in this study. In a series of chimeric and mutant P-glycoproteins, we have replaced discrete segments of NBD1 by their counterpart in NBD2 and have analyzed the effect of these substitutions on P-glycoprotein function. The first conclusion of these studies is based on results obtained with chimera F, which suggest that the two NBDs are not functionally equivalent and cannot be horizontally exchanged (duplication of NBD2 segment into NBD1) without loss of function. Two possible explanations can account for this finding. The first is that each NBD has its own particular function in the P-glycoprotein molecule or that both NBDs perform the same function, but within a specific environment and interactions provided by the sequence context of each half-molecule. It is interesting to note that these specific interactions, or different mechanistic aspects, are preserved among P-glycoproteins, since the vertical exchange of both NB sites (11; 22) between functionally distinct P-glycoproteins does not impair function(45) . A second possibility, which cannot be excluded from our analysis in LR73 cells, is that part of the phenotype of chimera F transfectants or transformants might be attributable to a decreased accumulation or altered targeting of the protein to the membrane. Indeed, Western blotting analysis of membrane fractions from yeast transformants expressing this protein showed a reduced accumulation of the chimeric protein compared to all other chimeras and mutant Mdr3 proteins which were expressed in membrane fractions at levels comparable to WT (data not shown).

The second and more definitive conclusion of this study is based on the analysis of chimeras and specific mutants, and shows that most of NBD1 can be replaced by corresponding NBD2 segments without any dramatic modification of the MDR phenotype conveyed by mdr3. However, replacement of a small number of residues near the Walker B motif, namely the ERGA segment (positions 522-525) and/or Thr-578, by the corresponding NBD2 residues, caused profound alterations of the drug resistance profile of P-glycoprotein, both in mammalian and yeast cells. In LR73 cells, these mutations resulted in a severe reduction (ACT) or almost complete loss (Adriamycin, COL) of resistance to some drugs while VBL resistance was retained (Fig. 4, E522D/R523K/A525T, T578C, and E522D/R523K/A525T/T578C). Alterations in drug resistance profiles, in general, and segregation of VBL resistance from Adriamycin and COL resistance, in particular, has previously been noted in other mutant P-glycoproteins. However, such P-glycoproteins usually carry mutations either within TM domains (TM11(30) ; TM4 and TM10(32) ) or in predicted intracellular loops intimately associated with the TM regions (34) , and in only one case has such a mutant phenotype been associated with mutations within NBDs(64) . The alteration of substrate specificity observed with the E522D/R523K/A525T and T578C mutants suggests that these NBD segments and residues are involved in substrate interaction, either directly or indirectly. Biochemical data in support of such a relationship include the observation that photoactivatable drug analogs label P-glycoprotein segments located immediately proximal to each NBD(27) . Also, in the case of another ABC family member, the CFTR protein, purified NBD1 by itself has chloride channel activity, suggesting that this NBD can play a direct role in substrate movement across the membrane(65) . Finally, genetic and biochemical analysis of the histidine permease of Salmonella typhimurium, revealed a strong association of the hisP nucleotide-binding component with the plasma membrane, and raised the possibility that a portion of hisP might actually span the membrane through specific interactions with the hisQ and hisM membrane components of the permease (reviewed in (39) ). It is also tempting to speculate that Thr-578 and ERGA(522-525) may participate in signal transduction between the NBDs and discrete TM-associated domains following either drug binding, ATP binding, or hydrolysis, since the latter domains have been shown to be primary drug-binding sites in P-glycoprotein (see Introduction). Biochemical studies of P-glycoprotein indeed suggest that conformational changes at both NBDs are important for P-glycoprotein function. For example, P-glycoprotein ATPase activity can be stimulated by certain drugs and modulators ( (43) and references cited within), suggesting that drug binding may induce conformational changes in the NBDs. Also, tryptophan fluorescence emission measurements from purified P-glycoprotein NBD2 indicate that ATP binding by itself is sufficient to induce major conformational changes in the NBD2 central region(66) . Finally, ATP hydrolysis at one or both NBDs must induce conformational changes (39) to mediate drug transport from the primary site of binding, the TM domains.

The highly conservative nature of the residues substituted in the E522D/R523K/A525T mutant protein indicates that even minor changes are not tolerated at these positions and cause severe alterations in transporter function (drug resistance and mating). The importance of these amino acid positions has also been confirmed for other ABC transporters. In hisP, a mutation at the Val residue (V152T) located at the position homologous to P-glycoprotein Ala-525 results in loss of function(67) , while in the ftsE cell division protein, a missense substitution (P135S) at a position homologous to P-glycoprotein Gly-524 induces thermo-sensitive filamentation(68) . The ERGA(NBD1)/DKGT(NBD2) P-glycoprotein segments are located immediately upstream of the highly conserved linker peptides preceding the B motifs. Linker peptides are characteristic of all ABC transporters and have indeed been speculated to mediate signal transduction between the TM domains and NBDs, on the basis of three-dimensional structure predictions(10, 40) .

The second deleterious substitution identified in this study is the T578C mutation (altered drug resistance profile and impaired mating). Thr-578 is immediately C-terminal to the B motif, in a region which is highly conserved between the two NBDs and which is, based on computer modelling studies, located at the periphery of the nucleotide-binding pocket of NBD1(40) . Interestingly, alignment of mdr3 and hisP in these regions(39, 67) , revealed that Thr-578 was located next to a hisP residue which is the site of a suppressor mutation (mutant 9087, position 205) that restores histidine transport in an otherwise deficient permease lacking the substrate-binding protein(69) . Thus, it has been proposed that this region of hisP may be implicated in signal transduction to the ATP binding/hydrolyzing subunit upon substrate binding to other portions of the transporter(70) . Therefore, a similar role in signal transduction may also be envisioned for Thr-578 in P-glycoprotein.

To examine whether mutations at the ERGA(522-525) and Thr-578 residues in NBD1 affect a mechanistic aspect of transport which is specific to P-glycoproteins or rather a more general mechanism common to other members of the ABC family, sequences of the corresponding NBD1 (Fig. 7A) and NBD2 (Fig. 7B) segments of the 2 human (h-Mdr1(8) , h-Mdr2(71) ) and 3 mouse (m-Mdr1(9) , m-Mdr2 (72) , and m-Mdr3(52) ) P-glycoproteins were aligned with those of other ABC transporters separated by large evolutionary distances. These included the fly (Drosophila Mdr49 and Mdr65(73) ), worm (Caenorhabditis elegans pgp-1(74) ), plant (Arabidopsis thaliana atpgp1(75) ), Plasmodium (pfmdr(13) ), and yeast (S. cerevisiae Ste6(57) ) homologs as well as the human CFTR (14) and Escherichia coli HlyB protein(76) . With respect to NBD1, both the ERGA and Thr-578 residues were conserved in all P-glycoproteins except for mouse mdr2 which showed a single E522D conservative substitution. This substitution does not play a key role, however, in potential functional differences among the different NBD1 of P-glycoprotein isoforms, since mdr2 NBD1 can complement the activity of mdr1 in conferring resistance to COL and Adriamycin(45) . The ERGA sequence was found conserved in many other ABC transporters, with 4 of the 8 sequences of the alignment showing identity at 3 out of 4 positions, while the Thr-578 was invariant in 6 of the 8 sequences (Fig. 7A). Therefore, these NBD1 residues seem to have been conserved throughout evolution, suggesting that they may participate in a common mechanistic aspect of transport, rather than in a P-glycoprotein-specific function. With respect to NBD2, both the DKGT sequence and Cys-1223 were invariant among the five P-glycoproteins. However, the DKGT sequence was not preserved in NBD2 of other ABC transporters (only 1 of 8 had 3 out of 4 identical residues), with some members showing amino acid deletions in this segment (MDR49, Ste6; Fig. 7B). Cys-1223 was retained in only 3 out of the 8 additional ABC transporters analyzed. This indicates that the DKGT segment and Cys-1223 may participate in a mechanistic aspect of transport that is specific to P-glycoprotein rather than common to other ABC transporters. Therefore, even though they differ only by conservative changes, the NBD1 ERGA and homologous NBD2 DKGT sequences appear to have been under different selective pressures during the evolution of the ABC family, ERGA retaining more of an ``ABC-type'' function while DKGT retained more of a ``P-glycoprotein type'' function. These different evolutionary pressures may reflect discrete functional differences between the two sequences which may help to explain why these two segments were identified as important for function in the experimental strategy adopted in our study (duplication of NBD2 segments into NBD1). By contrast, the central region of the NBDs separating the A and B motifs are not conserved among P-glycoproteins, nor among other ABC transporters (data not shown). Although one could have intuitively assigned to these segments the responsibility for functional differences between the two sites, the analysis of chimeras presented here clearly points to the opposite conclusion and indicates that these divergent segments are exchangeable without loss of function. This suggests that these sequences do not play a key catalytic role but may rather contribute to the structural organization of the NBD sites of P-glycoprotein.


Figure 7: Alignment of amino acid sequences from segments of nucleotide-binding domains of mouse (m-Mdr1, m-Mdr2, m-Mdr3) and human (h-MDR1, h-MDR2) P-glycoproteins and several other members of the ABC superfamily of transporters.



In this study, we analyzed the activity of chimeric and mutant P-glycoproteins in two different expression systems, a mammalian one in which the ability of P-glycoprotein to confer drug resistance was evaluated in LR73 cells (Table 3, Fig. 4), and a yeast one in which we measured the capacity of P-glycoprotein to confer cellular drug resistance and restore mating in a ste6 mutant strain (a-factor transport) (Fig. 6, Table 4). A comparison of the results obtained in these assays validates, in general, the use of the relatively new yeast expression system to study mammalian P-glycoproteins. High level P-glycoprotein expression in LR73 cells can only be achieved by the selection of stable transfectants in drug-containing medium. In the case of mutants affecting substrate specificity, the choice of drug used in the initial selection is critical since mutants with different levels of activity might compensate by a commensurate increase in expression of mutant proteins to correct for partial loss of function. Since all chimeras and mutants studied in this work displayed similar resistance to VBL (except chimera F), VBL selection could be performed without affecting resistance to the other drugs ( Fig. 4and Fig. 5). However, the study of mutants severly affected for different drugs of the MDR spectrum causes a problem that could be eliminated altogether in the yeast system where high levels of WT and mutant P-glycoprotein expression can be achieved in the absence of selection for P-glycoprotein function. For the chimeras and mutants presented in this study, a comparison of the drug resistance phenotypes of LR73 transfectants (Fig. 4) with the FK506 resistance of corresponding yeast transformants (Fig. 5C) shows an excellent quantitative correlation between FK506 resistance in yeast and ACT in LR73 cells. A similar correlation was also noted for COL and Adriamycin for all mutants except for chimera C which conveyed higher resistance to Adriamycin and COL than WT but lower resistance to FK506. Together, these results suggest that: 1) P-glycoprotein determinants involved in FK506/ACT resistance are similar, and are distinct from those involved in VBL resistance, and that 2) VBL preselection of LR73 transfectants did not bias the results of the drug resistance profile conferred by individual mutants. The co-segregation of ACT resistance (measured in LR73 cells) and FK506 resistance (measured in yeast) away from VBL resistance has been previously observed in P-glycoprotein mutants bearing mutations in TM11(30, 61) . The similar phenotype observed in TM11 and NBD1 mutants once again suggest that these two segments may be involved either directly or indirectly in FK506 but not in VBL transport.

Finally, the ability of WT and mutant P-glycoproteins to complement the STE6 gene and restore mating to a ste6 sterile strain was tested (Table 4). This assay is extremely sensitive and allows the quantitation of P-glycoprotein activity, as measured in mating frequencies, over a range of 4 orders of magnitude. This assay allows both the detection of subtle effects of mutations on P-glycoprotein function that may have gone otherwise unnoticed in the other assays, and the quantitation of low levels of residual P-glycoprotein activity in severely crippled mutants. The high sensitivity and wide analytical range of this assay identified in all chimeric and mutant P-glycoproteins tested (except chimera B and mutant N544Q) significant deviations from mating frequencies measured for WT mdr3. However, the relative mating activities measured were similar to FK506 resistance in yeast and ACT resistance in LR73 cells in individual mutants. Of particular interest were the results obtained with chimeras A and C which showed mating frequencies significantly greater than WT. Increased activity was also observed for these chimeras with respect to COL and Adriamycin resistance measured in LR73 cells. This suggests that exchange of certain fragments of NBD2 into NBD1 may release some negative regulation exerted at that site in the WT molecule. The only somewhat discordant results were those obtained with the E522D/R523K/A525T and T578C mutants which were completely inactive in mating while retaining partial FK506 resistance in yeast cells. These results indicate that the combined analysis of P-glycoprotein mediated cellular resistance to FK506 and STE6 complementation together provide accurate tools for the structure/function analysis of P-glycoprotein.

The identification of discrete segments (ERGA(522-525) and Thr-578) of NBD1 that cannot be substituted by the corresponding NBD2 sequences without alteration of either substrate specificity in LR73 cells or loss of STE6 complementation in yeast cells, has led to the hypothesis that these residues may be involved in signal transduction from the NBDs to the TM domains to effect transport. Although this proposition remains speculative, and although biochemical studies of these mutants have to be carried out to better understand the underlying defect (drug binding, ATPase activity), the power of genetic analysis in yeast will now allow the selection of intragenic suppressor mutations of the E522D/R523K/A525T and T578C mutants. These may in turn identify segments within P-glycoprotein that may interact with these residues, possibly shedding some light on intramolecular interactions in P-glycoprotein that are important for drug transport by this molecule.


FOOTNOTES

*
This work was supported in part by a grant (to P. G.) from the Medical Research Council of Canada. 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.

§
Supported by a fellowship from the Medical Research Council.

Supported by an E. W. R. Steacie fellowship from the Natural Sciences and Engineering Research Council of Canada and is an International Research Scholar of the Howard Hughes Medical Institute. To whom all correspondence should be addressed. Tel.: 514-398-7291; Fax: 514-398-7384; gros{at}medcor.mcgill.ca

The abbreviations used are: TM, transmembrane; NBD, nucleotide-binding domain; PCR, polymerase chain reaction; COL, colchicine; ACT, actinomycin D; VBL, vinblastine; WT, wild-type; CFTR, cystic fibrosis transmembrane conductance regulator.


ACKNOWLEDGEMENTS

We thank Dr. M. Raymond (Université de Montréal) for the gift of various yeast strains and for helpful discussions during this work, and T. Kwan for setting up the yeast growth inhibition assay.


REFERENCES

  1. Chan, H. S. L., Deboer, G., Thorner, P. S., Haddad, G., Gallie, B. L., and Ling, V. (1994)Hematol. Oncol. Clin. N.Am. 8, 383-410
  2. Gottesman, M. M., and Pastan, I.(1993)Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  3. Gros, P., and Buschman, E.(1993)Int. Rev. Cytol.137C,169-197
  4. Safa, A. R.(1992) Cancer Invest. 10, 295-305
  5. Cornwell, M. M., Tsuruo, T., Gottesman, M. M., and Pastan, I.(1987)FASEB J. 1, 51-54 [Abstract/Free Full Text]
  6. Schurr, E., Raymond, M., Bell, J. C., and Gros, P.(1989)Cancer Res. 49, 2729-2734 [Abstract]
  7. Ruetz, S., and Gros, P. (1994)J. Biol. Chem.269,12277-12284 [Abstract/Free Full Text]
  8. Chen, C., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B.(1986)Cell 47, 381-389 [Medline] [Order article via Infotrieve]
  9. Gros, P., Croop, J., and Housman, D.(1986)Cell 47, 371-380 [Medline] [Order article via Infotrieve]
  10. Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E., and Higgins, C. F. (1990)Nature 346, 362-365 [CrossRef][Medline] [Order article via Infotrieve]
  11. Higgins, F. C. (1992)Annu. Rev. Cell. Biol.8,67-113 [CrossRef]
  12. Kuchler, K., Sterne, R. E., and Thorner, J.(1989)EMBO J. 8, 3973-3984 [Abstract]
  13. Foote, S. J., Thompson, J. K., Cowman, A. F., and Kemp, D. J.(1989)Cell 57, 921-930 [Medline] [Order article via Infotrieve]
  14. Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S., and Tsui, L.-C.(1989)Science 245, 1066-1073 [Medline] [Order article via Infotrieve]
  15. Deverson, E. V., Gow, I. R., Coadwell, W. J., Monaco, J. J., Butcher, G. W., and Howard, J. C. (1990)Nature 348, 738-741 [CrossRef][Medline] [Order article via Infotrieve]
  16. Monaco, J. J., Cho, S., and Attaya, M.(1990)Science 250, 1723-1725 [Medline] [Order article via Infotrieve]
  17. Spies, T., Cerundolo, V., Colonna, M., Cresswell, P., Townsend, A., and DeMars, R. (1992)Nature 355, 644-646 [CrossRef][Medline] [Order article via Infotrieve]
  18. Trowsdale, J., Hanson, I., Mockridge, I., Beck, S., Townsend, A., and Kelly, A.(1990) Nature 348, 741-744 [CrossRef][Medline] [Order article via Infotrieve]
  19. Raymond, M., Gros, P., Whiteway, M., and Thomas, D. Y.(1992)Science 256, 232-234 [Medline] [Order article via Infotrieve]
  20. Ruetz, S., Raymond, M., and Gros, P.(1993)Proc. Natl. Acad. Sci. U. S.A.90,11588-11592 [Abstract]
  21. Simon, S. M., and Schindler, M.(1994)Proc. Natl. Acad. Sci. U. S. A. 91, 3497-3504 [Abstract]
  22. Ruetz, S., and Gros, P. (1994)Cell77,1071-1081 [Medline] [Order article via Infotrieve]
  23. Higgins, C. F. (1994)Cell79,393-395 [Medline] [Order article via Infotrieve]
  24. Raviv, Y., Pollard, H. B., Bruggeman, E. P., Pastan, I., and Gottesman, M. M.(1990) J. Biol. Chem. 265, 3975-3980 [Abstract/Free Full Text]
  25. Homolya, L., Holló, Z., Germann, U. A., Pastan, I., Gottesman, M. M., and Sarkadi, B.(1993)J. Biol. Chem. 268, 21493-21496 [Abstract/Free Full Text]
  26. Morris, D. I., Speicher, L. A., Ruoho, A. E., Tew, K. D., and Seamon, K. B.(1991) Bichemistry 30, 8371-8379
  27. Greenberger, L. M. (1993)J. Biol. Chem.268,11417-11425 [Abstract/Free Full Text]
  28. Choi, K., Chen, C.-J., Kriegler, M., and Roninson, I. B.(1988)Cell 53, 519-529 [Medline] [Order article via Infotrieve]
  29. Devine, S. E., Ling, V., and Melera, P. W.(1992)Proc. Natl. Acad. Sci. U.S.A.89,4564-4568 [Abstract]
  30. Dhir, R., Grizzuti, K., Kajiji, S., and Gros, P.(1993)Biochemistry 32, 9492-9499 [Medline] [Order article via Infotrieve]
  31. Gros, P., Dhir, R., Croop, J., and Talbot, F.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 7289-7293 [Abstract]
  32. Loo, T. W., and Clarke, D. M.(1993)J. Biol. Chem. 268, 3143-3149 [Abstract/Free Full Text]
  33. Loo, T. W., and Clarke, D. M.(1993)J. Biol. Chem. 268, 19965-19972 [Abstract/Free Full Text]
  34. Loo, T. W., and Clarke, D. M.(1994)J. Biol. Chem. 269, 7243-7248 [Abstract/Free Full Text]
  35. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J.(1991)Science 253, 202-205 [Medline] [Order article via Infotrieve]
  36. Powis, S. J., Deverson, E. V., Coadwell, W. J., Ciruela, A., Huskisson, N. S., Smith, H., Butcher, G. W., and Howard, J. C.(1992)Nature 357, 211-215 [CrossRef][Medline] [Order article via Infotrieve]
  37. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J.(1982)EMBO J. 1, 945-951 [Medline] [Order article via Infotrieve]
  38. Fry, D. C., Kuby, S. A., and Mildvan, A. S.(1986)Proc. Natl. Acad. Sci. U. S. A. 83, 907-911 [Abstract]
  39. Ames, G. F.-L., Mimura, C. S., Holbrook, S. R., and Shyamala, V.(1992)Adv. Enzymol. Areas Mol. Biol. 65, 1-47 [Medline] [Order article via Infotrieve]
  40. Mimura, C. S., Holbrook, S. R., and Ames, G. F.-L.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 84-88 [Abstract]
  41. Dano, K.(1973) Biochim. Biophys. Acta323,466-483 [Medline] [Order article via Infotrieve]
  42. Skovsgaard, T. (1978)Cancer Res.38,4722-4727 [Medline] [Order article via Infotrieve]
  43. Shapiro, A. B., and Ling, V.(1994)J. Biol. Chem. 269, 3745-3754 [Abstract/Free Full Text]
  44. Loo, T. W., and Clarke, D. M.(1994)J. Biol. Chem. 269, 7750-7755 [Abstract/Free Full Text]
  45. Buschman, E., and Gros, P.(1991)Mol. Cell. Biol. 11, 595-603 [Medline] [Order article via Infotrieve]
  46. Azzaria, M., Schurr, E., and Gros, P.(1989)Mol. Cell. Biol. 9, 5289-5297 [Medline] [Order article via Infotrieve]
  47. Tsui, L.-C.(1992) Trends Genet.8,392-398 [Medline] [Order article via Infotrieve]
  48. Berkower, C., and Michaelis, S.(1991)EMBO J. 10, 3777-3785 [Abstract]
  49. Sheppard, D. N., Ostedgaard, L. S., Rich, D. P., and Welsh, M. J.(1994) Cell 76, 1091-1098 [Medline] [Order article via Infotrieve]
  50. Anderson, M. P., and Welsh, M. J.(1992)Science 257, 1701-1704 [Medline] [Order article via Infotrieve]
  51. Smit, L. S., Wilkinson, D. J., Mansoura, M. K., and Collins, F. S.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9963-9967 [Abstract]
  52. Devault, A., and Gros, P.(1990)Mol. Cell. Biol. 10, 1652-1663 [Medline] [Order article via Infotrieve]
  53. Vernet, T., Dignard, D. and Thomas, D. Y.(1987)Gene (Amst.)52,225-233 [CrossRef][Medline] [Order article via Infotrieve]
  54. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego
  55. Southern, P. J., and Berg, P.(1982)J. Mol. Appl. Genet. 1, 327-341 [Medline] [Order article via Infotrieve]
  56. Tang-Wai, D. F., Brossi, A., Arnold, L. D., and Gros, P.(1993)Biochemistry 32, 6470-6476 [Medline] [Order article via Infotrieve]
  57. McGrath, J. P., and Varshavsky, A.(1989)Nature 340, 400-404 [CrossRef][Medline] [Order article via Infotrieve]
  58. Ito, H., Fokuda, K., Murata, K., and Kimura, A.(1983)J. Bacteriol. 153,163-168 [Medline] [Order article via Infotrieve]
  59. Sherman, F., Fink, G. R., and Hicks, J. B. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, NY
  60. Sprague, G. F. J. (1991)Methods Enzymol. 194, 77-93 [Medline] [Order article via Infotrieve]
  61. Raymond, M., Ruetz, S., Thomas, D. Y., and Gros, P.(1994)Mol. Cell. Biol. 14, 277-286 [Abstract]
  62. Buschman, E., Arceci, R. J., Croop, J. M., Che, M., Arias, I. M., Housman, D. E., and Gros, P. (1992)J. Biol. Chem. 267, 18093-18099 [Abstract/Free Full Text]
  63. Laemmli, U. K. (1970)Nature227,680-685 [Medline] [Order article via Infotrieve]
  64. Hoof, T., Demmer, A., Hadam, M. R., Riordan, J. R., and Tümmler, B.(1994) J. Biol. Chem. 269, 20575-20583 [Abstract/Free Full Text]
  65. Arispe, N., Rojas, E., Hartman, J., Sorscher, E. J., and Pollard, H. B.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1539-1543 [Abstract]
  66. Baubichon-Cortay, H., Baggett, L. G., Dayan, G., and Di Pietro, A.(1994)J. Biol. Chem. 269, 22983-22989 [Abstract/Free Full Text]
  67. Shyamala, V., Baichwal, V., Beall, E., and Ames, G. F.-L.(1991)J. Biol. Chem. 266, 18714-18719 [Abstract/Free Full Text]
  68. Gibbs, T. W., Gill, D. R., and Salmond, G. P. C.(1992)Mol. Gen. Genet. 234, 121-128 [Medline] [Order article via Infotrieve]
  69. Speiser, D. M., and Ames, G. F.-L.(1991)J. Bacteriol. 173, 1444-1451 [Medline] [Order article via Infotrieve]
  70. Petronilli, V., and Ames, G. F.-L.(1991)J. Biol. Chem. 266, 16293-16296 [Abstract/Free Full Text]
  71. Van der Bliek, A. M., Kooiman, P. M., Schneider, C., and Borst, P.(1988) Gene (Amst.)71,401-411 [CrossRef][Medline] [Order article via Infotrieve]
  72. Gros, P., Raymond, M., Bell, J., and Housman, D.(1988)Mol. Cell. Biol. 8, 2770-2778 [Medline] [Order article via Infotrieve]
  73. Wu, C. T., Budding, M., Griffin, M. S., and Croop, J. M.(1991)Mol. Cell. Biol. 11, 3940-3948 [Medline] [Order article via Infotrieve]
  74. Lincke, C. R., Smit, J. J. M., van der Velde-Koerts, T., and Borst, P.(1991) J. Biol. Chem. 266, 5303-5310 [Abstract/Free Full Text]
  75. Dudler, R., and Hertig, C.(1992)J. Biol. Chem. 267, 5882-5888 [Abstract/Free Full Text]
  76. Felmlee, T., Pellet, S., and Welch, R. A.(1985)J. Bacteriol.163,94-105 [Medline] [Order article via Infotrieve]

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