(Received for publication, April 5, 1995)
From the
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
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) 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
(GX 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 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.
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
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.
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
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
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
[
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.
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
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
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
Figure 4:
Multidrug resistance profiles of LR73
cells transfected with WT, chimeric, or mutant mdr3 cDNAs. D
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
Figure 5:
Drug resistance profiles of G418
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.
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
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 (1 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 Finally, the ability of WT and
mutant P-glycoproteins to complement the STE6 gene and restore
mating to a ste6 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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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) .
GKS/T) and Walker B
(R/KX
LHyd
D, 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
LSGGX
RHydXHydA(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) .
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.
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
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.
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.
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 (MATaste6
ura3) which
has been described previously(57) . Strain DC17 (MAT
his1) 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.
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).
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.
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.
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/KX
LHyd
D)
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).
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.
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) .
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.
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).
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.
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.
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).
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.
1; 2
2) 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).
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.