©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Membrane Topology of a Cysteine-less Mutant of Human P-glycoprotein (*)

(Received for publication, August 1, 1994; and in revised form, October 27, 1994 )

Tip W. Loo David M. Clarke (§)

From the Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.


INTRODUCTION

P-glycoprotein is a 170-kDa plasma membrane glycoprotein that is responsible for the phenomenon of multidrug resistance in mammalian cells (see reviews by Roninson(1991) and Gottesman and Pastan(1993)). The protein confers resistance to a broad range of cytotoxic agents that do not have a common structure or intracellular target; examples include anticancer drugs such as vinblastine and doxorubicin, antimicrotubule drugs such as colchicine and podophyllotoxin, and toxic peptides such as valinomycin and gramicidin D.

Human P-glycoprotein, encoded by the MDR1(^1)gene, consists of 1280 amino acids organized in two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids. Each repeat consists of an NH(2)-terminal hydrophobic domain containing six potential transmembrane sequences followed by a hydrophilic domain containing a nucleotide binding site. The amino acid sequence and domain organization of the protein is typical of the ABC (ATP-binding cassette) superfamily of transporters (Hyde et al., 1990).

Al-Shawi et al.(1994) showed that ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide. Maximal inactivation coincided with labeling at two sites, with approximately equal distribution of label between the NH(2)-terminal and COOH-terminal halves of the molecule. ATP prevented the inactivation of P-glycoprotein by N-ethylmaleimide. These results suggest that there is a critical sulfhydryl residue within each catalytic site. They predicted that the critical cysteines were located in the homology A sequences (GNSGCGKS and GSSGCGKS, respectively; Walker et al.(1982)) in the two predicted nucleotide binding domains of Chinese hamster P-glycoprotein.

In this study, we tested whether any of the cysteines, including those in the homology A regions, were critical for function by constructing a P-glycoprotein devoid of cysteines. The Cys-less P-glycoprotein retained the ability to confer resistance to cytotoxic agents. This finding provided the basis for an approach to studying the topology of P-glycoprotein. Our approach was to introduce cysteine residues into putative extracellular or cytoplasmic loops of Cys-less P-glycoprotein, followed by chemical modification with membrane-permeant or impermeant thiol-specific probes to determine their sidedness. We show that the pattern of labeling of the cysteine residues is consistent with a model which predicts that P-glycoprotein contains 12 transmembrane segments.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis

A full-length MDR1 cDNA cloned from a human kidney cortex cDNA library (Bell et al., 1986) and modified to encode the epitope for monoclonal antibody A52 (Zubrzycka-Gaarn et al., 1984) at the COOH-terminal end of the protein, was inserted into the mammalian expression vector pMT21, as described previously (Loo and Clarke, 1993). The sequence at the COOH terminus of P-glycoprotein that would normally end as TKRQ now became TKRAISLISNSCSPEFDDLPLAEQREACRRGDPRQ. Oligonucleotide-directed mutagenesis was carried out as described previously (Loo and Clarke, 1993).

Cell Culture

Procedures for transient transfection of human HEK 293 cells or stable transfection of mouse NIH 3T3 cells, followed by selection in the presence of vinblastine (5 nM) or colchicine (45 nM) have been described previously (Loo and Clarke, 1993). Drug sensitivity was determined by a tetrazolium-based assay for cell viability (Alley at al., 1988) as described previously (Loo and Clarke, 1993). A cell line stably expressing SERCA1 Ca-ATPase was generated by cotransfecting vectors p91023B SERCA1 (Clarke et al., 1989) and pWL-neo (Stratagene) into NIH 3T3 cells, followed by selection in the presence of G418 (Life Technologies, Inc.). Lysates of G418-resistant clones were screened with monoclonal antibody A52 to identify clones expressing SERCA1 Ca-ATPase.

Radiolabeling

Cells cultured on 60-mm diameter dishes were pulse-labeled for 20 min at 37 °C in methionine- and cysteine-free media with 102 µCi/ml TranS-label (1180 Ci/ml, ICN Biomedicals). The labeling medium was replaced with medium containing 10% (v/v) calf serum, and the cells incubated at 37 °C. At various intervals, the monolayers were washed twice with ice-cold PBS and cells collected by scraping. Immunoprecipitation with monoclonal antibody A52 was carried out as described previously (Loo and Clarke, 1993).

Labeling with Biotin Maleimide

Cells expressing the desired construct were washed three times with PBSCM (PBS containing 1 mM MgCl(2) and 0.1 mM CaCl(2)). For blocking, the cells were preincubated with stilbenedisulfonate maleimide (4-acetamido-4`-maleimidylstilbene-2,2`-disulfonic acid, Molecular Probes Inc.) in PBSCM at room temperature for 5-30 min, washed three times with PBSCM, and then biotinylated by incubation with biotin maleimide (3-(N-maleimidylpropionyl)biocytin, Molecular Probes Inc.) in PBSCM. Biotin maleimide was added from a stock prepared in Me(2)SO (dimethyl sulfoxide). The concentration of Me(2)SO in the labeling medium did not exceed 1% (v/v). After incubating for 5-30 min at room temperature, the cells were washed three times with PBSCM containing 2% (v/v) 2-mercaptoethanol, once with PBS, and then solubilized with lysis buffer. Insoluble material was removed by centrifugation at 15,000 times g for 15 min. The lysates were immunoprecipitated with monoclonal antibody A52. The immunoprecipitates were subjected to SDS-PAGE, transferred onto nitrocellulose, and biotinylated proteins detected using streptavidin-conjugated horseradish peroxidase and chemiluminescence (Amersham Corp.).


RESULTS

Mutagenesis of Individual Cysteine Residues

The modified human P-glycoprotein in this study contains 9 cysteine residues at positions 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304. The first objective was to test whether any individual cysteine was critical for structure or function. Accordingly, site-directed mutagenesis was used to construct mutant cDNAs in which each cysteine was changed to alanine. Mutant cDNAs were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. All of the mutants yielded a protein of apparent mass 170 kDa, and the relative resistances of each of these mutants to colchicine, vinblastine, doxorubicin, and actinomycin D were indistinguishable from that of wild-type P-glycoprotein-A52 (data not shown). These results indicate that none of the cysteine residues were essential for activity.

Construction and Activity of a Cys-less P-glycoprotein

Since none of the cysteines were essential for activity, we constructed a Cys-less P-glycoprotein-A52 for use in topology studies. Cysteine residues could then be reintroduced in the predicted cytoplasmic or extracellular loops connecting transmembrane segments of P-glycoprotein and reacted specifically with either permeant or impermeant sulfhydryl reactive compounds. Accordingly, a Cys-less mutant was constructed by simultaneously changing the codons of all cysteine residues to alanine, including those of the 2 cysteines (Cys-1288 and Cys-1304) flanking the epitope for monoclonal antibody A52. We have previously observed that mutation of the cysteine residues flanking the epitope for monoclonal antibody A52 does not affect the binding of monoclonal antibody A52 to this epitope. (^2)The ability of the Cys-less mutant to confer drug resistance was tested by transfecting drug-sensitive NIH 3T3 cells, followed by selection in the presence of vinblastine (5 nM) and colchicine (45 nM). Drug-resistant colonies were obtained in cells transfected with the cDNA coding for the Cys-less P-glycoprotein, whereas no drug-resistant colonies were obtained in cells transfected with vector alone. Several Cys-less clones were isolated and expanded. The ability of the Cys-less mutant to confer resistance was compared to that of wild-type P-glycoprotein-A52. As shown in Fig. 1, the Cys-less mutant had only slightly decreased relative resistance to vinblastine (0.70), colchicine (0.81), doxorubicin (0.78), and actinomycin D (0.63) relative to wild-type enzyme. These results suggest that the Cys-less mutant retains drug transport with only a small reduction in efficiency of the enzyme.


Figure 1: Comparison of relative resistances of cells expressing wild-type or Cys-less P-glycoprotein-A52 as a function of level of expression. Relative resistance was determined by dividing the LD (the drug concentration that inhibits plating efficiency by 50%) by the amount of P-glycoprotein-A52/1 times 10^5 cells. The amount of P-glycoprotein-A52 was determined as described previously (Loo and Clarke, 1993). The results are presented relative to the wild-type enzyme, which is arbitrarily assigned a value of 1. Wild-type, open histograms; Cys-less mutant, shaded histograms.



The biosyntheses of wild-type and Cys-less P-glycoprotein-A52s were studied in a pulse-chase assay (Fig. 2). Newly synthesized P-glycoprotein possesses three N-linked oligosaccharide chains and migrates with an apparent molecular mass of 150 kDa on SDS-PAGE gels (Fig. 2, time 0). After transit through the Golgi apparatus, P-glycoprotein migrates with an apparent molecular mass of 170 kDa, reflecting processing of its N-linked oligosaccharides (Fig. 2, time 6 h). A similar pattern was observed for the Cys-less mutant. At 0 h, the Cys-less P-glycoprotein-A52 had an apparent mass of 150 kDa. By 6 h, the major labeled product had an apparent mass of 170 kDa. Two differences, however, were observed. Firstly, processing of the wild-type enzyme to the mature (170 kDa) form occurred more rapidly than that of the Cys-less mutant. One hour after labeling, approximately equal amounts of the 150-kDa and 170-kDa forms of the enzyme were present. By contrast, less than 25% of the Cys-less mutant was processed to the 170-kDa enzyme after 1 h. A second difference was that the half-life of the mature (170 kDa) Cys-less protein was about 24 h compared to 48-72 h for wild-type P-glycoprotein-A52. These differences were observed in several clones, suggesting that the differences observed were not due to variabilities in the cell lines. Apparently, mutation of all cysteines to alanine resulted in a protein which was less stable than wild-type enzyme.


Figure 2: Kinetics of maturation of wild-type and Cys-less P-glycoprotein-A52. NIH 3T3 cells expressing wild-type or Cys-less P-glycoprotein-A52 were pulse-labeled with [S]Met and [S]Cys for 20 min at 37 °C and chased at the same temperature for 0-24 h. Cell lysates were immunoprecipitated with monoclonal antibody A52 and separated by SDS-PAGE followed by fluorography. 170 and 150 indicate the positions of the mature and core-glycosylated forms of P-glycoprotein-A52, respectively.



Topology Studies

The construction of a functional P-glycoprotein devoid of cysteine residues provided the basis of an approach to study the topology of the molecule. Using the cDNA coding for Cys-less P-glycoprotein-A52, we changed individual amino acids in the predicted cytoplasmic or extracellular loops to cysteine, which were then reacted with either membrane-permeant or impermeant sulfhydryl reagents in intact cells to determine their sidedness. The results of this approach are only valid if it can be shown that the mutants retain their native structure. The best assay for native structure is retention of function. Accordingly, we introduced cysteine residues into the cDNA of Cys-less P-glycoprotein-A52 and tested the ability of the mutant proteins to confer drug resistance in transfected cells (Table 1). Fifteen of the 19 cysteine mutant cDNAs yielded P-glycoproteins of apparent mass 170 kDa that conferred resistance to vinblastine and colchicine. No drug-resistant clones were obtained after transfection with mutants Lys-272 Cys, Ser-795 Cys, Asn-809 Cys, or Tyr-853 Cys. All of these mutations were found to affect biosynthesis of P-glycoprotein, since the major product in these mutants was a protein of apparent mass 150 kDa, when their cDNAs were transiently expressed in HEK 293 cells (data not shown). By contrast, all of the active mutants yielded a protein of apparent mass 170 kDa as the major product after transient transfection (data not shown).



The location of cysteine residues that were reintroduced in P-glycoprotein is shown in Fig. 3. Cysteine residues in the predicted extracellular loops should be accessible to membrane-impermeant thiol-specific reagents in intact cells. The cysteine residue of mutant Pro-745 Cys should be extracellular, since the epitope for monoclonal antibody MRK-16, which binds to an extracellular domain of P-glycoprotein, has been shown to consist of the region surrounding Pro-745, as well as residues between TM1 and TM2 (Georges et al., 1993). Similarly, Schinkel et al.(1993) showed that all three glycosylation sites in P-glycoprotein lie between predicted transmembrane segments TM1 and TM2, indicating that this region is exposed on the cell surface. Intact cells expressing mutant Pro-745 Cys or Cys-less P-glycoprotein-A52 were incubated with various concentrations of biotin maleimide (3-(N-maleimidylpropionyl)biocytin), which is a thiol-specific compound (Bayer et al., 1985). The maleimide compounds react specifically with thiol groups in P-glycoprotein-A52 since we were not able to detect reaction of N-[^3H]ethylmaleimide with the Cys-less mutant of P-glycoprotein-A52 (data not shown). Cells treated with biotin maleimide were solubilized and immunoprecipitated with monoclonal antibody A52. Fig. 4shows an immunoblot of the immunoprecipitated proteins developed with streptavidin-conjugated horseradish peroxidase and chemiluminescence. Labeling of mutant Pro-745 Cys was detected after treatment with 1 µM biotin maleimide, with maximal labeling occurring at a concentration of 10 µM. The reaction was specific, since no labeling of Cys-less P-glycoprotein-A52 was detected even when labeling was performed in the presence of 2 mM biotin maleimide. Labeling of mutant Pro-745 Cys was also rapid as the reaction was essentially complete within 2 min (data not shown). The permeability of biotin maleimide was tested by treating cells expressing mutant Cys-1304. This mutant is devoid of cysteines except for Cys-1304, which flanks the epitope for monoclonal antibody A52. This cysteine residue is at the COOH-terminal end of the second nucleotide-binding domain, and faces the cytoplasm (Yoshimura et al., 1989). In an immunohistochemical study, we found that reaction of monoclonal antibody A52 with P-glycoprotein-A52 requires permeabilization of cells,^2 suggesting that the epitope for monoclonal antibody is located in the cytoplasm. As shown in Fig. 4, substantial labeling of the mutant P-glycoprotein occurred when the cells were incubated in the presence of 1 mM biotin maleimide. This result suggests that biotin maleimide can cross the membrane and label internal cysteines when used at a relatively high concentration.


Figure 3: Location of introduced cysteines in the predicted structural model of P-glycoprotein. The model is that proposed by Juranka et al.(1989) and Gottesman and Pastan (1988). The positions of the introduced cysteine residues are indicated by largecircles. The results of labeling with biotin maleimide and membrane-impermeant stilbenedisulfonate maleimide are indicated: solid black circles, labeled with 20 µM biotin maleimide after incubation for 5 min and labeling was blocked by preincubation with stilbenedisulfonate maleimide; dotted circles, labeling required incubation for 30 min in the presence of 200 µM biotin maleimide and labeling was not blocked by preincubation with stilbenedisulfonate maleimide; open circles, no labeling under either condition; striped circles, inactive mutants.




Figure 4: Labeling of Cys-less P-glycoprotein mutants containing an introduced cysteine on the extracellular (Pro-745 Cys) or cytosolic side (Cys-1304) of the plasma membrane with biotin maleimide. NIH 3T3 cells expressing Cys-less, Pro-745 Cys or Cys-1304 mutants of P-glycoprotein-A52 were washed three times with PBSCM and incubated for 5 min at room temperature, in the presence of varying concentrations (0-1000 µM) of biotin maleimide. The cells were washed with PBSCM containing 2% (v/v) 2-mercaptoethanol, solubilized, and immunoprecipitated with monoclonal antibody A52. The immunoprecipitates were subjected to SDS-PAGE and biotinylated proteins detected using streptavidin-conjugated horseradish peroxidase and chemiluminescence. The concentration of biotin maleimide (µM) and positions of the mature (170 kDa) P-glycoprotein-A52 are indicated.



Stilbene disulfonate maleimide (4-acetamido-4`-maleimidylstilbene-2,2`-disulfonic acid) is a membrane-impermeable maleimide. It possesses two charged sulfonate groups, but unlike biotin maleimide, which must first be dissolved in dimethyl sulfoxide, it is freely soluble in water. When cells expressing mutants Pro-745 * Cys or Cys-1304 were treated with stilbenedisulfonate maleimide before incubation with biotin maleimide, biotinylation of only mutant Pro-745 Cys was blocked (Fig. 5). These results are consistent with Pro-745 being located on the extracellular surface, since it is rapidly biotinylated in the presence of low concentrations of biotin maleimide and biotinylation is blocked when the cells are preincubated with membrane-impermeant stilbenedisulfonate maleimide. By contrast, biotinylation of mutant Cys-1304 required a relatively higher concentration (200 µM) of biotin maleimide and biotinylation was not blocked by the membrane-impermeant maleimide (Fig. 5). These properties suggest that Cys-1304 is located in the cytoplasm. To confirm that these properties indicate a cytoplasmic orientation, labeling was carried out on SERCA1 Ca-ATPase which is found in the endoplasmic reticulum in transfected cells (Maruyama et al., 1989), with its nucleotide-binding domain (containing 13 cysteine residues) facing the cytoplasm. As shown in Fig. 5, the labeling of Ca-ATPase was similar to that of mutant Cys-1304, in that biotinylation only occurred at a relatively high concentration (>100 µM) of biotin maleimide and was not blocked by pretreatment of the cells with stilbenedisulfonate maleimide.


Figure 5: Inhibition of biotinylation with stilbenedisulfonate maleimide. NIH 3T3 cells expressing mutant Pro-745 Cys were incubated in the absence(-) or presence (+) of 20 µM stilbenedisulfonate maleimide for 5 min at room temperature, washed three times with PBSCM, and then incubated for 5 min with PBSCM containing 20 µM biotin maleimide. Cells expressing Cys-1304 or SERCA1 Ca-ATPase were incubated in the absence(-) or presence (+) of 200 µM stilbenedisulfonate maleimide for 30 min at room temperature, washed three times with PBSCM, and then incubated with PBSCM containing 200 µM biotin maleimide for 30 min. Biotinylated P-glycoprotein-A52 was detected as described in Fig. 4. The positions of P-glycoprotein-A52 (P-gp) and Ca-ATPase are indicated.



Labeling of other Cys mutants with stilbenedisulfonate maleimide and biotin maleimide was then done. In these cases, the mutants were expressed transiently in HEK 293 cells. In this expression system, transfected cells yield a high level of core-glycosylated P-glycoprotein (150 kDa), located in the endoplasmic reticulum, as well as mature P-glycoprotein (170 kDa) (Loo and Clarke, 1994a). This enabled us to examine the labeling pattern of P-glycoprotein located intracellularly (150 kDa) as well as the mature form (170 kDa) located in the plasma membrane. Labeling was first carried out using a low concentration of biotin maleimide (20 µM) and a relatively short incubation period (5 min): conditions that favor biotinylation of extracellular thiol groups. As shown in Fig. 6(panelB), biotinylation of the mature 170-kDa form of P-glycoprotein was observed for mutants Gly-324 Cys, Pro-745 Cys, and Lys-967 Cys. These results are consistent with the predicted location of these residues on the extracellular surface (Fig. 3). Labeling was then carried out at a relatively high concentration (200 µM) of biotin maleimide and for a longer period (30 min). Under these conditions, both the 150-kDa and 170-kDa forms of P-glycoprotein of mutants Thr-173 Cys, Asn-280 Cys, Ser-831 Cys, Ser-931 Cys, and Cys-1304 were biotinylated. By contrast, only the mature 170-kDa form of mutants Gly-324 Cys, Pro-745 Cys, and Lys-967 Cys were labeled (Fig. 6, panel C). This difference can be explained on the basis of their location inside or outside the lumen of the endoplasmic reticulum during biosynthesis of the 150-kDa intermediate. Cysteine residues that are predicted to reside on the extracellular surface would be located in the lumen of the endoplasmic reticulum during biosynthesis, while those predicted to be eventually intracellular would also be exposed to the cytoplasm in the core-glycosylated intermediate. Since the cysteine residues of mutants Thr-173 Cys, Asn-280 Cys, Ser-831 Cys, Ser-931 Cys, and Cys-1304 are predicted to be intracellular in the mature enzyme, both 150-kDa and 170-kDa forms of the enzyme would be expected to be labeled and not blocked by pretreatment with stilbenedisulfonate maleimide (Fig. 6, panelD). On the other hand, the cysteine residues in the mature form (170 kDa) of mutants Gly-324 Cys, Pro-745 Cys, and Lys-967 Cys are predicted to be extracellular and, therefore, would be expected to be found within the lumen of the endoplasmic reticulum in the 150-kDa form of the enzyme. Therefore, labeling of these cysteine residues would require the probe to traverse two membranes, resulting in only a minor amount of the enzyme (150 kDa) being labeled. Biotinylation of the 150-kDa form of the enzyme of these mutants required the use of even higher concentrations (2 mM) of biotin maleimide (data not shown). Consistent with the interpretation that the cysteine residue of mutants Gly-324 Cys, Pro-745 Cys, and Lys-967 Cys are located on the extracellular surface (and within the lumen of the endoplasmic reticulum) was the finding that biotinylation of the mature P-glycoprotein was blocked by pretreatment with membrane-impermeant stilbenedisulfonate maleimide (Fig. 6, panel D).


Figure 6: Labeling of active P-glycoprotein Cys mutants. HEK 293 cells were transfected with cDNAs coding for Cys-less P-glycoproteins containing introduced cysteine residues. Forty-eight hours after transfection, the cells were washed three times with PBSCM and then incubated for 5 min in the presence of 20 µM biotin maleimide (panelB) or for 30 min in the presence of 200 µM biotin maleimide before (panelC) or after preincubation (panel D) for 30 min in the presence of 200 µM stilbenedisulfonate maleimide. The cells were washed with PBSCM containing 2% (v/v) 2-mercaptoethanol, solubilized, and immunoprecipitated with monoclonal antibody A52. The immunoprecipitates were subjected to SDS-PAGE and transferred onto nitrocellulose, and biotinylated P-glycoprotein-A52 was detected using streptavidin-conjugated horseradish peroxidase and chemiluminescence. A sample of whole cell lysate was also subjected to immunoblot analysis with monoclonal antibody A52 and horseradish peroxidase conjugated goat anti-mouse antibody, followed by chemiluminescence (panel A). 170 and 150 indicate the positions of the mature and core-glycosylated forms of P-glycoprotein-A52, respectively.



Biotinylation of the cysteine residue of mutants Thr-209 Cys, Gly-211 Cys, Thr-215 Cys, Ser-238 Cys, Ser-850 Cys, Gly-854 Cys, or Trp-855 Cys was not detected, suggesting that these cysteine residues were likely inaccessible. Six of the inaccessible cysteines, located at positions 209, 211, 215, 850, 854, and 855, were predicted to lie on the extracellular surface between TM3 and TM4 or TM9 and TM10, while Cys-238, located between TM3 and TM4, is predicted to be cytoplasmic. The loops connecting segments TM3 and TM4, and TM9 and TM10 are predicted to be short, consisting of 6 and 1 amino acids, respectively. Therefore, there may be little or no protrusion of these loops into the extracellular space, thus preventing reaction of the cysteine residues in these locations (positions 209, 211, 215, 850, 854, and 855) with biotin maleimide. We were, however, able to biotinylate the cysteine residues in these positions (including Cys-238) after denaturation with SDS (data not shown).


DISCUSSION

The results in this study show that a mutant P-glycoprotein, which is devoid of cysteine residues, retains the ability to confer resistance to cytotoxic drugs. Apparently, none of the cysteines play a direct role in the mechanism of drug transport. These results suggest that inactivation of P-glycoprotein by thiol-specific reagents such as N-ethylmaleimide (Al-Shawi et al., 1994) is probably due to the introduction of one or more bulky groups into the molecule, rather than due to the removal of a critical sulfhydryl group. Cysteines, however, do appear to contribute to folding and stability of P-glycoprotein. Maturation of the Cys-less P-glycoprotein was slower than wild-type enzyme, and the relative half-life of the mature mutant enzyme was shorter than that of wild-type enzyme.

The Cys-less mutant of P-glycoprotein was used to study the topology of the protein in the membrane. Site-directed mutagenesis was used to reintroduce cysteine residues into the loops between the transmembrane segments of the cysteine-less P-glycoprotein, which were then probed with membrane-permeant or impermeant thiol-specific reagents. The advantage of this approach is that the assays are performed on functional molecules in intact cells. Using this approach, we found that the regions between predicted transmembrane segments TM5 and TM6, TM7 and TM8, and TM11 and TM12 to be extracytoplasmic, as predicted in the current model of P-glycoprotein (Fig. 3).

The loops between transmembrane segments TM3 and TM4, as well as TM9 and TM10 are predicted to be extracellular (Fig. 3). Introduction of cysteine residues into these loops, however, resulted in no detectable biotinylation. These loops are predicted to be short (6 amino acids and 1 amino acid, respectively) and may not protrude significantly above the lipid bilayer, thereby making them sterically inaccessible to labeling with biotin maleimide. Recently, Skach and Lingappa(1994) showed that the region between transmembrane segment TM3 and TM4 of human P-glycoprotein is indeed extracellular. They investigated the topology of TM3 and TM4 using protease protection of a defined reporter epitope, which was expressed as a fusion protein with segments of MDR1 in Xenopus laevis oocytes. They found that TM3 and TM4 spanned the membrane in the orientation predicted by the hydropathy-based model (Fig. 3).

Cysteine residues introduced into predicted cytoplasmic loops between transmembrane segments TM2 and TM3 (Thr-173 Cys), TM4 and TM5 (Asn-280 Cys), TM8 and TM9 (Ser-831 Cys), and TM11 and TM12 (Ser-931 Cys) exhibited labeling properties consistent with this location. Labeling of these cysteine residues (as well as Cys-1304 in the COOH terminus of the nucleotide binding domain) required relatively high concentrations (200 µM) of biotin maleimide and longer incubation periods (30 min). Consistent with their cytoplasmic location was the finding that biotinylation of these residues was not blocked by pretreatment with membrane-impermeant maleimide.

Our results suggest that the region between predicted transmembrane TM8 and TM9 is cytoplasmic. This is in contrast to the results of Skach et al.(1993). Again, using fusion proteins expressed in Xenopus oocytes and a cell-free translation system, they found that the region between TM8 and TM9 resides in the lumen of the endoplasmic reticulum (extracytosolic), and that Asn-809 (within predicted TM8 and TM9) was glycosylated. This is in contrast to our findings that the region between TM8 and TM9 (Ser-831 Cys) is in the cytoplasm and that the COOH-terminal half of the molecule is not glycosylated. In a previous study (Loo and Clarke, 1994b), we showed that expression of the COOH-terminal half of P-glycoprotein as a separate polypeptide in NIH 3T3, HEK 293, or insect Sf9 cells results in a functional molecule which is not glycosylated. The absence of glycosylation in this region has also been demonstrated in a photolabeling study on MDR1 expressed in mammalian cells (Bruggemann et al., 1989). Glycosylation of the COOH-terminal half of P-glycoprotein, however, was also observed when expression was carried out in a cell-free system (Zhang and Ling, 1991). These discrepancies may reflect differences between in vivo and in vitro expression of P-glycoprotein. It is also possible that truncations and additions of a passenger domain to P-glycoprotein in the study by Skach et al.(1993) could result in misfolding of the the molecule. Biosynthesis and maturation of P-glycoprotein can be particularly sensitive to even single amino acid changes in the predicted cytoplasmic loops (Loo and Clarke, 1994a). The segment of amino acids encompassing TM7 to TM9 is particularly sensitive to perturbations. Mutations to 9 residues within or immediately NH(2)-terminal to TM7 (Loo and Clarke, 1994c) and to 10 of 27 residues between TM8 and TM9^2 result in the synthesis of only 150-kDa core-glycosylated P-glycoprotein, which is trapped in the endoplasmic reticulum. These mutants may be retained in the endoplasmic reticulum due to misfolding.

This study shows that a cysteine-less mutant can be a useful tool for studying the topology of P-glycoprotein and may be a useful approach for studying the topology of other membrane proteins. Cysteine-less mutants could also be used to study the static and dynamic aspects of structure and function of membrane-bound proteins as proposed by van Iwaarden et al.(1991). Single Cys mutants can be tagged with reactive spin labels or fluorescent probes after purification and studied spectroscopically after reconstitution into lipid bilayers. Distances between extracellular loops could be estimated by using thiol-specific cross-linkers of various lengths. We are currently using these approaches to gain insight into the structure-function relationships of human P-glycoprotein.


FOOTNOTES

*
This research was supported by a grant (to D. M. C.) as part of a group grant 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.

§
Scholar of the Medical Research Council of Canada. To whom correspondence and reprint requests should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1105; Fax: 416-978-8765.

(^1)
The abbreviations used are: MDR, multidrug resistance; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane segment; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; stilbenedisulfonate maleimide, 4-acetamido-4`-maleimidylstilbene-2,2`-disulfonic acid; SERCA, sarcoendoplasmic reticulum Ca-ATPase.

(^2)
D. M. Clarke and T. W. Loo, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Dr. David H. MacLennan for the cDNA encoding SERCA1 Ca-ATPase, the A52 epitope, and monoclonal antibody used in this study. We thank Dr. Randal Kaufman (Genetics Institute, Boston, MA) for pMT21. We appreciate the gift of HEK 293 cells from Dr. David Hampson (Department of Pharmacy, University of Toronto).


REFERENCES

  1. Alley, M. C., Scudiero, D. A., Monks, A., Hursey, M. L., Czerwinski, M. J., Fine, D. L., Abbott, B. J., Mayo, J. G., Shoemaker, R. H., and Boyd, M. R. (1988) Cancer Res. 48, 589-601 [Abstract]
  2. Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992 [Abstract/Free Full Text]
  3. Bayer, A. E., Zalis, M. G., and Wilchek, M. (1985) Anal. Biochem. 149, 529-536 [Medline] [Order article via Infotrieve]
  4. Bell, G. I., Fong, N. M., Stempien, M. H., Warmsted, M. A., Caput, D., Ku, L, Urdes, M. S., Rall, L. B., and Sanchez-Pescador, R. (1986) Nucleic Acids Res. 14, 8427-8446 [Abstract]
  5. Bruggemann, E. P., Germann, U. A., Gottesman, M. M., and Pastan, I. (1989) J. Biol. Chem. 264, 15483-15488 [Abstract/Free Full Text]
  6. Clarke, D. M., Maruyama, K., Loo, T. W., Leberer, E., Inesi, G., and MacLennan, D. H. (1989) J. Biol. Chem. 264, 11246-11251 [Abstract/Free Full Text]
  7. Georges, E., Tsuruo, T., and Ling, V. (1993) J. Biol. Chem. 268, 1792-1798 [Abstract/Free Full Text]
  8. Gottesman, M. M., and Pastan, I. (1988) J. Biol. Chem. 263, 12163-12166 [Free Full Text]
  9. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  10. Hyde, S. C. Emsley, P., Hartshorn, M. J., Mimmack, M. L., 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. Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592 [Abstract/Free Full Text]
  12. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 3143-3149 [Abstract/Free Full Text]
  13. Loo, T. W., and Clarke, D. M. (1994a) J. Biol. Chem. 269, 7243-7248 [Abstract/Free Full Text]
  14. Loo, T. W., and Clarke, D. M. (1994b) J. Biol. Chem. 269, 7750-7755 [Abstract/Free Full Text]
  15. Loo, T. W., and Clarke, D. M. (1994c) J. Biol. Chem. 269, 28683-28689 [Abstract/Free Full Text]
  16. Maruyama, K., Clarke, D. M., Fujii, J., Loo, T. W., and MacLennan, D. H. (1989) Cell Motil. Cytoskel. 14, 26-34 [Medline] [Order article via Infotrieve]
  17. Roninson, I. B. (ed) (1991) Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells , Plenum Press, New York
  18. Schinkel, A. F., Kemp, S., Dolle, M., Rudenko, G., and Wagenaar, E. (1993) J. Biol. Chem. 268, 7474-7481 [Abstract/Free Full Text]
  19. Skach, W. R., and Lingappa, V. R. (1994) Cancer Res. 54, 3202-3209 [Abstract]
  20. Skach, W. R., Cayalag, M. C., and Lingappa, V. R. (1993) J. Biol. Chem. 268, 6903-6908 [Abstract/Free Full Text]
  21. van Iwaarden, P. R., Pastore, J. C., Konings, W. N., and Kaback, H. R. (1991) Biochemistry 30, 9595-9600 [Medline] [Order article via Infotrieve]
  22. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951 [Medline] [Order article via Infotrieve]
  23. Yoshimura, A., Kuwazura, Y., Sumizawa, T., Ichikawa, M., Ikeda, S. I., Ueda, T., and Akiyama, S. I. (1989) J. Biol. Chem. 264, 16282-16291 [Abstract/Free Full Text]
  24. Zhang, J.-T., and Ling, V. (1991) J. Biol. Chem. 266, 18224-18232 [Abstract/Free Full Text]
  25. Zubrzycka-Gaarn, E., MacDonald, G., Phillips, L., Jorgensen, A. O., and MacLennan, D. H. (1984) J. Bioenerg. Biomembr. 16, 441-446 [Medline] [Order article via Infotrieve]

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