(Received for publication, May 8, 1995; and in revised form, July 7, 1995)
From the
P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains interact in P-glycoprotein, we expressed each domain as a separate polypeptide and tested for associations using coimmunoprecipitation assays. We found that the interactions between the two halves of P-glycoprotein were mediated through associations between the two transmembrane domains as well as through the nucleotide-binding domains. In addition, the nucleotide-binding domain also associated with the transmembrane domain in each half of the molecule. By contrast, we could not detect any association either between the first nucleotide-binding domain and the second transmembrane domain, or between the second nucleotide-binding domain and the first transmembrane domain. We then tested whether individual domains associated with molecular chaperones, since biogenesis of P-glycoprotein appears to involve the chaperones calnexin and Hsc70. We found that calnexin associated only with the transmembrane domains, while Hsc70 associated only with the nucleotide-binding domains. These results suggest that noncovalent interaction between the domains of P-glycoprotein can contribute to structure and function of P-glycoprotein and that chaperones may participate in the folding of each domain.
P-glycoprotein, the product of the human MDR1 gene, is an energy-dependent transport protein which interacts with a wide variety of hydrophobic cytotoxic agents that do not have a common structure or intracellular target (see reviews by Roninson(1991) and Gottesman and Pastan(1993)). The protein is clinically important since it contributes to the phenomenon of multidrug resistance during chemotherapy of human cancers (see reviews by Endicott and Ling(1989), Van der Bliek and Borst(1992), and Gottesman and Pastan(1993)).
P-glycoprotein
consists of 1280 amino acids organized in two tandem repeats of 610
amino acids, joined by a linker region of 60 amino acids (Chen et
al., 1986). Each repeat consists of an NH-terminal
hydrophobic domain containing six potential transmembrane sequences
followed by a hydrophilic domain containing a nucleotide binding site.
Studies on the topology suggest that each transmembrane domain (TMD) (
)consists of six membrane-spanning helices (Loo and Clarke,
1995). Genetic (Choi et al., 1988; Gros et al., 1991;
Devine et al., 1992; Loo and Clarke, 1993a, 1993b, 1994a) and
biochemical analyses (Bruggemann et al., 1989, 1992;
Greenberger, 1993; Zhang et al., 1995a) indicate that residues
within the TMD contribute to drug binding. The hydrophilic domains
containing the consensus nucleotide binding folds (NBF) have been found
to bind ATP (Azzaria et al., 1989; Baubichon-Cortay et
al., 1994).
Functional studies suggest that different domains of P-glycoprotein must interact with each other for transport to occur. It has been demonstrated that ATPase activity is stimulated in the presence of drug substrates (Ambudkar et al., 1992; Sarkadi et al., 1992; Sharom et al., 1993; Al-Shawi and Senior, 1993; Shapiro and Ling, 1994). Coupling of ATPase activity to drug binding involves interactions between both homologous halves of the molecule since no drug-stimulated ATPase activity was observed when each half was expressed as a separate polypeptide (Loo and Clarke, 1994b).
The physical basis for interactions between domains is not known. Such interactions have been shown to be critical for function in bacterial ABC transporters, where individual domains are composed of separate polypeptides. In the histidine permease, for example, noncovalent interactions between all four domains were required to form a functional complex (Kerppola et al., 1991). In this study, we tested whether there are physical interactions between specific domains of P-glycoprotein by expressing each domain as a separate polypeptide and using coimmunoprecipitation assays. In addition, we tested whether chaperones may participate in the folding of the individual domains as there is evidence that they play a role in biosynthesis of the enzyme (Loo and Clarke, 1994c). We found that interactions between the homologous halves of the molecule are mediated by association between the TMDs as well as between NBFs. Specific associations were also observed between TMDs and NBFs. In addition, the molecular chaperone calnexin is involved in the folding of the TMDs, whereas Hsc70 participates in the folding of NBFs.
In order to express TMD1 (residues 1-379)
containing the epitope for monoclonal antibody A52, pBSGMDR1-A52 was
digested with BstBI (nt 1133) and HindIII (nt 3843),
filled-in with Klenow and the vector fragment ligated with T DNA ligase.
The cDNA coding for TMD2 (residues 681-1025) and
containing the epitope for monoclonal antibody A52 was created by
digesting the cDNA coding for the COOH-terminal half-molecule of
P-glycoprotein, pBSMDR1A52NH
(Loo and Clarke, 1994b)
with StuI (nt 3072) and EcoRV (nt 3849), and ligation
of the vector fragment.
The cDNA coding for NBF1-A52 was obtained by
ligating the cDNAs coding for residues 1-16 (EcoRI (nt
-76) to DraI (nt 48)), residues 388-529 (SspI (nt 1159) to ApaI (nt 1582)) and the epitope
for monoclonal antibody A52 (ApaI (nt 1582) to XhoI
(nt 4061)). The ApaI to XhoI fragment containing the
A52 epitopes was isolated from pBSGMDR1A52COOH (Loo and Clarke,
1994b). The fragment coding for NBF1-A52 was subcloned into pMT21. In
some experiments, NBF1 lacking the epitope for monoclonal antibody A52
was needed. The cDNA coding for NBF1 was obtained by ligating the cDNA
coding for residues 1-16 with that coding for residues
388-682 (SspI (nt 1159) to XhoI (nt 4061))
isolated from pBSGMDR1
COOH.
The cDNA coding for NBF2-A52 was
constructed by ligating the fragment coding for residues 1025 to 1311
(StuI (nt 3072) to XhoI (nt 4061) to
pBSGMDR1A52NH
which had been linearized with HindIII, filled-in with Klenow fragment, followed by digestion
with XhoI.
In some instances, NBF2 was fused to calbindin D-28K. Fusion of NBF2 to calbindin (residues 19 to 261) (Parmentier et al., 1987) allowed us to differentiate NBF2 from TMD2 in SDS-PAGE.
The fragments coding for each domain of P-glycoprotein were subcloned into the EcoRI and XhoI sites of the expression vector, pMT21 and the nucleotide sequence confirmed by sequencing (Sanger et al., 1977).
Expression and Glycosylation- Fig. 1shows the four domains of P-glycoprotein and the composition of the constructs used to express each domain as a separate polypeptide. In most cases, the epitope for monoclonal antibody A52 was added at the COOH terminus of each construct to facilitate identification of the expressed mutant proteins, as well as for immunoprecipitation. We have previously found that the addition of an epitope tag to the full-length protein (Loo and Clarke, 1993a), or to either half-molecule forms of P-glycoprotein did not inhibit function (Loo and Clarke, 1994b).
Figure 1:
Model of human
P-glycoprotein and linear representation of individual domains. A, a simplified model of P-glycoprotein indicating the
location of the predicted glycosylation sites (Y), the linker region
(&cjs0467;), and the individual domains. TM1-TM12 correspond to the predicted transmembrane helices. B, a
linear representation of P-glycoprotein, showing insertion of a segment
(in bold) containing the epitope for monoclonal antibody A52 (underlined). C, the NH-terminal
transmembrane domain (TMD1); D, the
NH
-terminal nucleotide-binding domain (NBF1); E, the COOH-terminal transmembrane domain (TMD2), and F, the COOH-terminal nucleotide-binding domain (NBF2). All constructs contain the epitope for monoclonal
antibody A52 (underlined).
Fig. 2shows an
immunoblot of whole cell extracts of HEK 293 cells expressing various
domains of P-glycoprotein. Cells transfected with the cDNAs coding for
the NH-terminal (NBF1) and COOH-terminal (NBF2)
nucleotide-binding domains, expressed polypeptides with apparent masses
of 41 and 35 kDa, respectively, which are comparable to their predicted
sizes. By contrast, both the NH
-terminal (TMD1) and the
COOH-terminal (TMD2) transmembrane domains had apparent masses that
were smaller than that predicted from their cDNA when analyzed by
SDS-PAGE. TMD1 had an apparent mass of 39 kDa (unglycosylated, see
below), compared to its predicted size of 44 kDa, while TMD2 had an
apparent mass of 35 kDa, which is smaller that its predicted size of 40
kDa. The discrepancies in the apparent masses of the transmembrane
domains is probably due to incomplete denaturation, since the samples
were not heated after solubilization in SDS sample buffer. Heating of
these samples, but not NBF1 or NBF2, caused aggregation of these
polypeptides such that they remained in the stacking gel.
Figure 2:
Expression of the domains of
P-glycoprotein and sensitivity to endoglycosidase H digestion. HEK 293
cells transfected with vector alone (Control) or the cDNAs
coding for various domains were solubilized with buffer containing 50
mM sodium citrate, pH 5.5, 0.5% (w/v) SDS, 10 mM EDTA, and 1% (v/v) 2-mercaptoethanol. Each sample was divided into
two equal portions and incubated with (+) or without(-)
endoglycosidase H (1000 units, New England Biolabs) for 15
min at room temperature. The digestion was stopped by addition of an
equal volume of buffer containing 0.25 M Tris-HCl, pH 6.8, 4%
(w/v) SDS, 4% (v/v) 2-mercaptoethanol, and 20% (v/v) glycerol. The
reaction mixtures were subjected to SDS-PAGE and immunoblot analysis
with monoclonal antibody A52, as described under ``Experimental
Procedures.''
To determine if the expressed domains were inserted into membranes of HEK 293 cells, membranes were prepared from cells transfected with the cDNA coding for each domain and then treated with sodium carbonate. Carbonate extraction of membranes removes all but integral membrane proteins (Fujiki et al., 1982). It was found that wild-type P-glycoprotein-A52 and the TMD1 and TMD2 polypeptides were almost exclusively recovered in the pellet fractions, whereas NBF1 and NBF2 were present only in the supernatant fractions (data not shown). These results suggest that both TMD1 and TMD2 contain signals necessary for insertion into the membranes of the endoplasmic reticulum.
Human MDR1 contains three glycosylated sites in the first extracellular loop (Schinkel et al., 1993). Accordingly, we tested the sensitivity of the domain polypeptides to endoglycosidase H digestion. Fig. 2shows that the apparent molecular mass of TMD1 decreased from 46 kDa to approximately 39 kDa following endoglycosidase H treatment. By contrast, the TMD2, NBF1, or NBF2 were not sensitive to endoglycosidase H (Fig. 2) or N-glycanase F digestion (data not shown). These results suggest that only TMD1 was core-glycosylated and likely resides in the endoplasmic reticulum of transfected cells.
Figure 3:
Coimmunoprecipitation of domains of
P-glycoprotein. HEK 293 cells were transfected with the cDNAs coding
for various domains of P-glycoprotein. 30 h after transfection, the
cells were solubilized with 1% (w/v) digitonin. After centrifugation at
16,000 g for 15 min at 4 °C, the supernatant
fractions were immunoprecipitated with various antibodies as described
below. The immunoprecipitated proteins were separated by SDS-PAGE and
subjected to immunoblot analysis with a specific antibody against the
desired domain and developed using enhanced chemiluminescence (Amersham
Corp.). Panel A, cells transfected with the cDNA coding for
NH
-terminal half-molecule with no epitope for monoclonal
antibody A52, or cotransfected with the cDNA coding for the
COOH-terminal half-molecule containing the epitope for monoclonal
antibody A52, were immunoprecipitated with monoclonal antibody A52 and
the immunoblot developed with an affinity-purified rabbit polyclonal
antibody specific for the NH
-terminal nucleotide-binding
domain (NBF1). Panel B, cells transfected with cDNA
coding for TMD2 and containing the epitope for monoclonal antibody A52
or cotransfected with the cDNA coding for the NH
-terminal
half-molecule with no epitope for monoclonal antibody A52 were
immunoprecipitated with an affinity-purified rabbit polyclonal antibody
against NBF1 and the immunoblot developed with monoclonal antibody A52. Panel C, cells were transfected with the cDNA coding for NBF2
fused to calbindin or cotransfected with cDNA coding for NBF1 and
containing the epitope for monoclonal antibody A52 were
immunoprecipitated with monoclonal antibody A52 and the immunoblot
developed with a monoclonal antibody against calbindin (Sigma). Panel D, cells were transfected with the cDNA coding for
either TMD1 or TMD2 and containing the epitope for monoclonal antibody
A52 or cotransfected with the cDNA coding for NBF1 lacking the epitope
for monoclonal antibody A52, were immunoprecipitated with an
affinity-purified rabbit polyclonal antibody against NBF1 and the
immunoblot developed with monoclonal antibody A52. Panel E,
cells were transfected with the cDNA coding for either NBF1, TMD1, or
TMD2 and containing the epitope for monoclonal antibody A52, or
cotransfected with the cDNA coding for NBF2 which was fused to
calbindin, were immunoprecipitated with anti-calbindin monoclonal
antibody and the immunoblot developed with monoclonal antibody A52. The
positions of the NH
-terminal half-molecule (N-Half), the nucleotide-binding (NBF1, NBF2) and the transmembrane domains (TMD1, TMD2) are indicated.
To identify the regions of
interaction between the two halves of P-glycoprotein, we coexpressed
the various domains (Fig. 1) of P-glycoprotein followed by
immunoprecipitation with specific antibodies. Fig. 3B shows that TMD2 was recovered in the immune complex using an
affinity-purified rabbit polyclonal antibody against NBF1, when
coexpressed with the NH-terminal half-molecule. In the
absence of the NH
-terminal half-molecule, however, the
antibody did not immunoprecipitate TMD2 (Fig. 3B).
These results indicate that one possible site of interaction between
the two half-molecules of P-glycoprotein is through the transmembrane
domains.
To determine whether the cytoplasmic domains were also responsible for association of the two halves of P-glycoprotein, we coexpressed NBF1 containing an epitope for monoclonal antibody A52 together with NBF2 which had been fused to calbindin D-28K. Fig. 2shows that TMD2 and NBF2 containing the A52 tags have approximately the same mass in SDS-PAGE. By fusing NBF2 to calbindin, the molecular mass of NBF2 is increased by approximately 25 kDa (see below), allowing it to be distinguished from TMD2 in SDS-PAGE.
Fig. 3C shows that NBF2-calbindin could be recovered with monoclonal antibody A52, only in the presence of NBF1. Similarly, when NBF1 is coexpressed with NBF2-calbindin and immunoprecipitated with monoclonal antibody against calbindin, NBF1 could also be immunoprecipitated (Fig. 3E). These results indicate that interaction between the two nucleotide-binding domains could also contribute to physical association between the two half-molecules of P-glycoprotein.
Another potentially important association in P-glycoprotein is through association of the nucleotide-binding domains with the transmembrane domains. The observation that ATPase activity is stimulated by drug binding suggests a functional interaction between the cytoplasmic and transmembrane domains. Fig. 3D shows that, when NBF1 was coexpressed with either TMD1 or TMD2 and immunoprecipitated with antibody against NBF1, only TMD1 was coimmunoprecipitated. One possible explanation that TMD2 was not coimmunoprecipitated was that it was not expressed in the cells. We confirmed, however, that both NBF1 and TMD2 were indeed coexpressed by performing an immunoblot analysis on a sample of cells transfected with the cDNAs coding for both domains prior to immunoprecipitation studies (Fig. 4). These results suggest that NBF1 can associate with TMD1 but not with TMD2. Similarly, we attempted to identify associations of NBF2 with the transmembrane domains. Fig. 3E shows that there was association of NBF2 with TMD2 but not with TMD1. The large amount of immunoreactive material of apparent mass between 50 and 100 kDa is due to reaction of the anti-mouse secondary antibody with the mouse monoclonal antibody (anti-calbindin D) that was used during the immunoprecipitation. The immunoblot in Fig. 3E was exposed to film for a very short period of time compared to that in Fig. 4, to prevent masking of any signal by the immunoreactive material of 50-100 kDa. Therefore, the presence of a signal (Fig. 3E, lanes 2 and 4, respectively), suggests that these are significant associations. Extraction of cells with SDS (Fig. 4) or with digitonin (data not shown) resulted in the majority of TMD1 as well as the other domains being present in the soluble fraction.
Figure 4: Expression of cDNAs coding for individual domains or pairs of domains of P-glycoprotein in HEK 293 cells. HEK 293 cells were transfected with cDNA coding for an individual domain or pairs of domains of P-glycoprotein, solubilized with 2% (w/v) SDS and the cell extracts subjected to SDS-PAGE and immunoblot analysis using an affinity-purified rabbit polyclonal antibody specific to NBF1 (N) or with a mixture of monoclonal antibodies against the A52 epitope and calbindin (A52/Cal), as described under ``Experimental Procedures.'' The positions of the nucleotide-binding (NBF1, NBF2) and transmembrane domains (TMD1, TMD2) are indicated.
These results show that there are physical interactions between the transmembrane and nucleotide-binding domains in each half of P-glycoprotein.
Figure 5:
Association of Hsc70 with full-length
wild-type and mutant (Pro
Gly) P-glycoproteins.
HEK 293 cells expressing wild-type or mutant (Pro
Gly) P-glycoproteins-A52 were solubilized with 1% (w/v) digitonin.
After centrifugation at 16,000
g for 15 min at 4
°C, the supernatant fractions were immunoprecipitated with anti
Hsc70 (B) or with monoclonal antibody A52 (C). The
immunoprecipitates were subjected to SDS-PAGE and immunoblot analysis
with monoclonal antibody A52 (B) or with anti-Hsc70 monoclonal
antibody (C). A sample of the supernatant fractions (A) was subjected to immunoblot analysis with monoclonal
antibody A52. Bands migrating at 170 and 150 kDa correspond to mature
and core-glycosylated forms of P-glycoprotein, respectively. The
positions of antibody (Ab) and Hsc70 are indicated. Incompletely
denatured antibody can be seen in B because these samples were
denatured at room temperature, whereas samples in C were
heated for 2 min at 100 °C prior to
SDS-PAGE.
Fig. 6shows that Hsc70 associates with both half-molecules of
P-glycoprotein. This association is likely mediated through physical
interaction with the nucleotide-binding domains since it was found that
Hsc70 was present in the immune complexes from cells transfected with
cDNAs coding for NBF1 or NBF2 and immunoprecipitated with monoclonal
antibody A52, but not in the immune complexes from cells expressing
TMD1 or TMD2. In addition, immunoprecipitation with anti-Hsc70
antibodies showed that NBF1 or NBF2 were recovered in the immune
complexes but not TMD1 or TMD2 (Fig. 7). The SERCA1
Ca-ATPase of sarcoplasmic reticulum was included as a
control because it already contains the epitope for monoclonal antibody
A52, and is localized to the endoplasmic reticulum in transfected
mammalian cells (Maruyama et al., 1989). No Hsc70 was detected
in the immune complexes from cells transfected with the cDNA coding for
SERCA1 Ca
-ATPase and immunoprecipitated with
monoclonal antibody A52 (Fig. 6). These results suggest that
Hsc70 associations are specific.
Figure 6:
Association of Hsc70 with various domains
of P-glycoprotein. HEK 293 cells transfected with vector alone (Control), with cDNA coding for various domains of
P-glycoprotein and containing the epitope for monoclonal antibody A52
or with cDNA coding for SERCA1 Ca-ATPase were
solubilized with 1% (w/v) digitonin. After centrifugation, the
supernatants were immunoprecipitated with monoclonal antibody A52. The
immune complexes were subjected to SDS-PAGE and immunoblot analysis
with anti-Hsc70 monoclonal antibody as described under
``Experimental Procedures.'' A sample of whole cell extract
from control cells (Cell Extract) which was not subjected to
immunoprecipitation was also immunoblotted. The positions of Hsc70 and
antibody (IgG H) are indicated.
Figure 7: Domains of P-glycoprotein immunoprecipitated with Hsc70. HEK 293 cells expressing the various domains of P-glycoprotein and containing the epitope for monoclonal antibody A52 or vector alone (Control) were solubilized with 1% (w/v) digitonin and the supernatant fraction immunoprecipitated with anti-Hsc70 monoclonal antibody. The immune complexes were subjected to SDS-PAGE and immunoblot analysis with monoclonal antibody A52. The position of NBF1 and NBF2 are indicated.
A similar approach was used to test
for association of calnexin with each domain of P-glycoprotein. Fig. 8shows that calnexin also associates with both
half-molecules. The association with P-glycoprotein appears to be
mediated through the transmembrane domains, since it was detected only
in the immune complexes of the TMD1 and TMD2 but not in those of the
nucleotide-binding domains. Similarly, when cell extracts of
transfected cells were immunoprecipitated with anti-calnexin
antibodies, followed by detection with monoclonal antibody A52, only
the transmembrane domains were recovered (Fig. 9). The
association also appeared to be specific since no detectable calnexin
was found in the immune complexes from cells transfected with the cDNA
coding for SERCA1 Ca-ATPase (Fig. 8). These
results show that calnexin associates with transmembrane domains,
whereas Hsc70 associates with the nucleotide binding domains.
Figure 8:
Association of calnexin with various
domains of P-glycoprotein. HEK 293 cells expressing vector alone (Control), various domains of P-glycoprotein and containing
the epitope for monoclonal antibody A52 or SERCA1
Ca-ATPase were solubilized with 1% (w/v) digitonin
and the supernatant fractions immunoprecipitated with monoclonal
antibody A52. The immune complexes were subjected to SDS-PAGE and
immunoblot analysis with affinity-purified rabbit anti-calnexin
antibody. A sample of whole cell extract from control cells (Cell
Extract) which was not subjected to immunoprecipitation was also
immunoblotted. The positions of calnexin and antibody (IgG H)
are indicated.
Figure 9: Domains of P-glycoprotein immunoprecipitated with anti-calnexin antibodies. HEK 293 cells expressing vector alone, or expressing individual domains of P-glycoprotein containing the epitope for monoclonal antibody A52, were solubilized with 1% (w/v) digitonin and the supernatant fractions immunoprecipitated with affinity-purified anti-calnexin antibody. The immune complexes were subjected to SDS-PAGE and immunoblot analysis with monoclonal antibody A52. The positions of antibody (Ab) and the transmembrane domains (TMD1, TMD2) are indicated.
Potential problems in a coimmunoprecipitation approach to
study protein interactions are nonspecific associations and aggregation
after lysis of the cells. To overcome these problems, incubations in
the presence of antibody were for short periods (2 h). In addition,
controls containing only one domain were run in parallel (Fig. 3). In each case, nonspecific aggregation was not
observed. Indeed, we found that the associations between domains
decreased with longer incubation periods (18 h). The associations were
specific since there were no associations between NBF1 and TMD2 or
between NBF2 and TMD1. Similarly, no associations were found between
SERCA1 Ca-ATPase and either NBF1, NBF2 or the
NH
-half molecule (data not shown). These results indicate
that there are specific noncovalent associations between the various
domains of P-glycoprotein.
An association between the two transmembrane domains was expected based on the results of photolabeling studies on P-glycoprotein. P-glycoprotein labeled with photoactive drug analogs followed by protease digestion revealed that the labeled fragments were from transmembrane domains from both homologous halves of P-glycoprotein (Bruggemann et al., 1989; 1992; Greenberger et al., 1991; Greenberger, 1993). About 50% of the label was found in each half of the molecule. In the presence of vinblastine, labeling of both halves was inhibited to a similar extent (Bruggemann et al., 1992). These results suggest that the labeling site is formed by both transmembrane domains.
An association between the nucleotide-binding domains, however, was rather surprising since there is no evidence of cooperativity in the ATPase activity of P-glycoprotein (Shapiro and Ling, 1994). Both nucleotide-binding domains appear to be capable of hydrolytic activity since low levels of ATPase activity could be detected when each half of P-glycoprotein was expressed as an individual polypeptides (Loo and Clarke, 1994b).
An association was also observed between the nucleotide-binding domains and the transmembrane domains. These interactions may be especially important for coupling of ATPase activity to drug binding, and could be mediated through the large cytoplasmic loops connecting the transmembrane segments. A number of mutations have been identified in these loops which alter the drug resistance profiles conferred by P-glycoprotein (Choi et al., 1988; Currier et al., 1992; Loo and Clarke, 1994a) and alter the pattern of coupling of drug binding to ATPase activity (Rao, 1995). Associations among residues within the four domains provide a mechanism for coupling of drug binding to ATPase activity, which is likely to be a key feature of drug transport.
Biosynthesis of the individual
domains appears to be mediated through interactions of molecular
chaperones. Association of calnexin with TMD1 is not surprising since
this chaperone transiently associates with a wide variety of
glycosylated proteins that are exported or targeted to the cell surface
(reviewed by Bergeron et al.(1994)). TMD2, however, was also
found to associate with calnexin, although we found no evidence that
this domain was glycosylated. Other unglycosylated proteins have been
identified which interact with calnexin, such as the nonglycosylated
-subunit of the T-cell receptor (Rajagopalan et al.,
1994). In addition, calnexin remains bound to the class I major
histocompatibility complex molecules after removal of oligosaccharides
by endoglycosidase H. Initiation of binding by calnexin to the class I
complex, however, was recently shown to require the presence of
oligosaccharides (Zhang et al., 1995b). Apparently calnexin
recognizes both carbohydrate and protein determinants of selected
proteins.
The molecular chaperone Hsc70 specifically associated with the nucleotide-binding domains. This class of chaperones has been implicated in stabilizing newly synthesized polypeptides, in mediating assembly of multimeric protein complexes, and in facilitating translocation of polypeptides across membranes (see reviews by Gething and Sambrook(1992), McKay(1993), and Becker and Craig(1994). Although members of the Hsp70/Hsc70 chaperone family are highly conserved, they show common and divergent specificities (Fourie et al., 1994). In general, they bind to a segment of the protein containing at least 7 residues that include large hydrophobic and basic amino acids with few or no acidic residues. The binding motif is best described as HyXHyXHyXHy, where Hy is a large hydrophobic or aromatic amino acid and X is any amino acid. There are several candidate regions in the nucleotide-binding domains of P-glycoprotein. For example, potential regions involved in interacting with Hsc/Hsp70 include residues 397-403 (VHFSYPS) in NBF1 and residues 1040-1046 (VVFNYPT) in the corresponding region of NBF2.
Association of each domain with at least one molecular chaperone
suggests that folding of P-glycoprotein is carefully monitored for
fidelity. This may account for retention in the endoplasmic reticulum
of a large number of mutant P-glycoproteins in which only a single
amino acid has been substituted (Loo and Clarke, 1993a, 1993b, 1994a,
1995). ()Elucidation of the sites of association between
molecular chaperones and P-glycoprotein would help in understanding the
role of the chaperones in the maturation of P-glycoprotein to form a
functional transporter.