(Received for publication, August 1, 1994; and in revised form, October 27, 1994 )
From the
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.
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()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
-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-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.
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
10
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.
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-[
H]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,
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).
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
-terminal to TM7 (Loo and Clarke, 1994c) and to 10 of 27
residues between TM8 and TM9
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.