From the
Recent studies reveal that the organization of the multidrug
resistance protein (Mdr) in the membrane is probably not exactly as
predicted from hydropathy profiling. When expressed in Escherichia
coli, phoA gene fusions can be utilized to study the
membrane topology of Mdr. Using this approach, it was proposed recently
that the N-terminal hydrophobic domain of Mdr spans the membrane six
times, in a different fashion from that predicted by hydropathy
analysis (Bibi, E. and Béj, O. (1994) J. Biol. Chem. 269, 19910-19915). In this study, we analyze
[Abstract]
mdr-phoA fusions constructed in the C-terminal half of Mdr. Overall, the
results presented here lead to a significant revision in the membrane
topology model of the C-terminal half of Mdr. The new topology is
discussed with regard to the hydropathy profiles of the well
characterized ABC proteins MalG and MalF, which are strikingly similar
to those of the N- and C-terminal halves of Mdr, respectively.
Multidrug resistance protein (Mdr)
A general secondary structure model for Mdr has been deduced from
hydropathy analysis. Presumably, Mdr contains 12 transmembrane segments
(TM) with the C and the N termini, as well as both nucleotide binding
domains, on the cytoplasmic surface of the membrane (Gottesman and
Pastan, 1988). The two halves of the molecule are connected by a highly
charged linker. General features regarding the orientation of Mdr have
been elucidated using sequence-specific antibodies (Kartner et
al., 1985, Yoshimura et al., 1989), but the detailed
topology of the protein in the membrane is still elusive. Experimental
analysis of the predicted membrane topology is important in order to
facilitate genetic and biochemical studies of Mdr function. In this
regard, recent studies (Zhang and Ling, 1991; Zhang et al.,
1993; Skach et al., 1993, Bibi and Béj, 1994) indicate
that the topology of Mdr in the membrane may differ from the predicted
structure.
In previous studies, it was shown that mouse Mdr1 (Gros
et al., 1986) can be expressed in E. coli in a
functional state and at a relatively high level (Bibi et al.,
1993). This opened the possibility of utilizing the well characterized
prokaryotic gene fusion approach (reviewed by Traxler et al. (1993)) to study the topology of Mdr. In a recent report (Bibi and
Béj, 1994) the heterologous expression system was used to
analyze the topology of the N-terminal hydrophobic domain of Mdr by
gene fusions with alkaline phosphatase as a reporter (Manoil and
Beckwith, 1985). In this study, we present an analysis of Mdr-alkaline
phosphatase fusions constructed in the C-terminal half of Mdr. The
results suggest that the homologous halves of Mdr are asymmetric in the
membrane.
A major deviation from the predicted structure of Mdr has
been readily observed when reporter polypeptides fused to the putative
extracellular loop following TM7 fail to cross the membrane (Skach
et al., 1993). Using the C-terminal 142-amino acid residues of
bovine prolactin as a reporter in Xenopus oocytes or in in
vitro expression systems, Skach et al.(1993) constructed
and analyzed Mdr-prolactin hybrids in which the prolactin derived
reporter was attached to amino acid residues 733 and 750, both located
in the C-terminal half of human MDR1 (see Fig. 5A for
the specific location of the fusions in the original topology model).
We constructed Mdr-alkaline phosphatase hybrids at Val
Although topological data for most members of
the ABC superfamily are not available, detailed studies have been
carried out with the maltose periplasmic permease (see, for example,
Prinz and Beckwith(1994); Boyd et al., 1993). The maltose
transport system of E. coli is composed of two integral
membrane proteins (MalG and MalF), a peripheral ATP binding subunit
(MalK), and a periplasmic component (maltose-binding protein). Although
the membrane components of the maltose transport system do not exhibit
significant homology at the amino acid level with the membrane domains
of Mdr, a striking similarity in their hydropathy profiles is apparent
(Fig. 4). The greatest similarity exists between the hydropathy
profiles of the N terminus of MalG and the N-terminal membrane domain
of Mdr. In addition, the hydropathy profile of the N-terminal portion
of MalF is similar to the respective region in the C-terminal membrane
domain of Mdr (Fig. 4). Extensive study of MalF topology, reveals
three TMs in this region of the protein (Boyd et al., 1993).
Importantly, the results presented here indicate that the analogous
portion of Mdr also contains three TMs (Fig. 5). The similar
organization in both systems may be attributed to the role of the
N-terminal sequences in membrane targeting and insertion. Such a role
for the N-terminal sequence of the C-terminal membrane domain of Mdr
was recently suggested when mutations in this region were shown to
abolish proper processing and targeting (Loo and Clarke, 1994).
We are grateful to Dr. H. Ronald Kaback and Dr. Steve
Karlish for helpful discussions during this work and to Dr. H. Ronald
Kaback for his critical reading of the manuscript. We thank Dr. Ophry
Pines for his valuable advice during the preparation of this manuscript
and Hadassa Waterman for her assistance during the rotation studies.
(
)
is a
complex integral membrane protein which plays a major role in drug
resistance of tumors (reviewed by Endicott and Ling(1989); Gottesman
and Pastan, 1993; Gros and Buschman, 1993). Mdr is a 170-kDa membrane
protein which belongs to the ABC (Hyde et al., 1990) or
traffic ATPase (Mimura et al., 1991) superfamily of transport
proteins. Proteins in this family contain two homologous nucleotide
binding domains and two integral membrane domains each containing six
putative transmembrane segments in
-helical conformation. Included
are the cystic fibrosis transmembrane conductance regulator (Riordan
et al., 1989), the yeast mating factor exporter (Ste6)
(Kuchler et al., 1989; McGrath and Varshavsky, 1989), the
hemolysin exporter (HlyB) (Felmlee et al., 1985), and many
so-called periplasmic permeases in Gram-negative bacteria, such as the
maltose transport system in Escherichia coli (Nikaido, 1994).
and Cys
, with the reporter following putative TM7
and two more hybrids at Thr
and Lys
, where
the alkaline phosphatase moiety is attached to residues preceding TM7.
In the E. coli system, the intracellular orientation of the
hybrids is judged by the fact that all of them exhibit low specific
alkaline phosphatase activities that are estimated from the absolute
activity and the level of expression as detected by immunoprecipitation
using antibodies against alkaline phosphatase (Fig. 1A).
The low activities indicate cytoplasmic disposition of the fused
reporters, despite the existence of a relatively long hydrophobic amino
acid residues stretch between Lys
and Val
(proposed TM7). Interestingly, in both the prokaryotic and the
eukaryotic systems, the reporters fail to cross the membrane, even
though they are located in the putative extracellular loop following
TM7. Skach et al. (1993) suggested that TM7 by itself is not
able to cross the membrane and requires topological information located
in the following transmembrane segments. However, careful examination
of the hydropathy profile of Mdr in this region
(Fig. 2A) reveals a stretch of relatively hydrophilic
amino acid residues in the middle of TM7 and raises the possibility
that TM7 (Fig. 2B) could cross the membrane twice
(Fig. 2C), with a short extracellular loop (composed of
Asn
, Gly
, Cys
, see
Fig. 5B) between the two membrane-spanning segments. To
study this possibility, we constructed additional hybrids, at
Gly
and Pro
which are located in the
vicinity of the short hydrophilic segment and at Leu
which is located in the N-terminal portion of TM7. The activities
and the immunoprecipitation analysis of this group of hybrids are
presented in Fig. 1B and demonstrate that hybrid
Leu
(low alkaline phosphatase activity) and hybrids
Gly
or Pro
(high alkaline phosphatase
activity) are potential borders for the first transmembrane segment.
Consequently, the second half of the original TM7 also crosses the
membrane as indicated by the flanking active (Pro
) and
non active (Val
and Cys
) hybrids
(Fig. 1B). Therefore, the results obtained with this set
of fusions fit the new proposed arrangement of Mdr.
Figure 5:
Secondary structure of the region
containing TMs 7, 8, and 9 of Mdr as proposed in this study
(B) and a previous model (A). The single amino acid
code is used; hydrophobic TMs are shown in vertical boxes.
Mdr-alkaline phosphatase hybrids are indicated. Positively and
negatively charged residues are slightly
shaded.
Figure 1:
Right panels, immunoprecipitation with
anti-alkaline phosphatase antibodies of
[S]methionine-labeled UT5600 cells expressing
various Mdr-alkaline phosphatase hybrids. Left panels,
specific activities measured and calculated for the same Mdr-alkaline
phosphatase hybrids as in the right panels. The same
transformants were used for immunoprecipitation and alkaline
phosphatase activity assays in each set (A, B, C, D). All the
methods used for the preparation of the gene fusion constructs (the
complete list of the synthetic oligodeoxynucleotides used in this study
is available upon request), the immunoprecipitation, and the alkaline
phosphatase activity assays were described in detail in Bibi and
Béj (1994).
Figure 2:
Hydropathy plot and the deduced secondary
structure model of the C-terminal transmembrane domain of mouse Mdr1.
A, the average local hydrophobicity at each residue calculated
by the method of Kyte and Doolittle (1982) is plotted on the
vertical axis versus the residue number on the horizontal
axis. Higher values represent greater hydrophobicity. The figure
is adopted from the output of the program DNA Strider. B,
secondary structure model according to Gottesman and Pastan (1988).
C, secondary structure model proposed in this study. The
arrow indicates the orientation of the TM (N to C
terminus)
To further
examine the results, we constructed a ``sandwich hybrid''
(Ehrmann et al., 1990), SanV717, between amino acid residues
Val and Asn
(Ile
was deleted
due to the gene manipulation). The alkaline phosphatase inserted at
this point is expected to be extracellular (Fig. 5B) if
the insertion does not damage the structure of Mdr. However, as shown
in Fig. 1D, the normalized activity of SanV717 is low
relative to a previously described sandwich fusion that was constructed
in an extracellular loop but in the N-terminal half of Mdr (SanL226,
Bibi and Béj, 1994). As expected, full-length Mdr-alkaline
phosphatase hybrid Ser
also exhibits low alkaline
phosphatase activity (Fig. 1D) consistent with its
cytoplasmic orientation. These observations thus indicate that unlike
with the simple hybrids (Gly
and Pro
), when
alkaline phosphatase is inserted as a sandwich near the short
hydrophilic loop, it is not fully translocated to the periplasmic
space. One possible explanation for the low activity is that the
alkaline phosphatase insert destroys the functional assembly of Mdr,
because the loop containing the insert is too short and may not be able
to promote correct organization with such a long polypeptide insert. If
this is the case, it is anticipated that the transport activity
mediated by Mdr containing such an insert will be drastically affected.
Accordingly, we compared tetraphenylarsonium (TPA
)
transport in cells expressing the sandwich hybrids SanV717 and SanL226
(Bibi and Béj, 1994), to cells expressing full-length
Mdr-alkaline phosphatase hybrid (Ser
) or native Mdr.
Mdr-mediated TPA
transport causes a lower level of
accumulation of the substrate (Bibi et al., 1993). In contrast
to SanL226, which exhibits wild-type activity as judged by the low
level of TPA
accumulation, SanV717 is completely
inactive (Fig. 3). The low transport activity mediated by
sandwich hybrid SanV717 is not due to low level of expression as
demonstrated by a semiquantitative immunoblot analysis were the
expression level of SanV717 was found similar to that of native Mdr,
but much higher than SanL226 (data not shown).
Figure 3:
Transport of TPA by
E. coli UT5600 harboring pT7-5, pTSanL226, pTSanV717,
pTS1276, or pTmdr1 (Bibi et al., 1993). Cells were
grown, induced with
isopropyl-1-thio-
-D-galactopyranoside, and treated with
EDTA as described in Bibi et al. (1993). Transport of
[
H]TPA (23.8 mCi/mmol) at a final concentration
of 0.4 mM was assayed at 34 °C by rapid
filtration.
The notion that TM7
is actually two transmembrane segments suggests a major change in the
orientation of the following transmembrane segment (indicated by an
arrow in Fig. 2). To examine this possibility, we
constructed hybrids in which alkaline phosphatase is fused to Mdr at
residues Ala and Tyr
in a region following
the next hydrophobic segment (Fig. 5). As shown in
Fig. 1C, hybrids Ala
and Tyr
exhibit high levels of normalized alkaline phosphatase
activities, clearly suggesting that the next transmembrane segment
flanked by amino acid residues Cys
and Ala
has an opposite orientation from the predicted structure and
further supporting the proposal that the original TM7 is in fact
composed of two transmembrane segments. These results are consistent
with those obtained with a Mdr-prolactin hybrid in which prolactin is
connected to amino acid residue 816 following TM8 (Skach et
al., 1993). In that construct, prolactin is detected in the lumen
of the microsomal membranes, in agreement with the results presented
here and with the notion that TM8 crosses the membrane but in an
opposite orientation.
Figure 4:
Comparison between the hydropathy profiles
of the membrane components of the maltose transport system and Mdr. See
the legend to Fig. 2 for details.
Although our proposal that TM7 consists of two TMs is supported by
results presented both here and with other experimental systems (Skach
et al., 1993; Zhang and Ling, 1991), it is inconsistent with
epitope mapping studies using monoclonal antibodies (mAb) MRK-16
(Georges et al., 1993) and MM4.17 (Cianfriglia et
al., 1994). Using these mAbs, it was proposed that a stretch of
amino acid residues 740-747 is extracellular. An explanation for
the discrepancy is not readily obvious, but may be provided in a recent
report by Jachez et al.(1994). In their study they demonstrate
that the extracellular binding levels of the MM4.17/MRK-16 epitope with
the mAbs is temperature-dependent (4-fold higher at 37 °C than at 4
°C), unlike the binding properties of another cell surface epitope
that is temperature-independent (MC57). In this regard, we speculate
that unlike the interaction with antibodies, gene fusion experiments
provide topological information at the time it is created
cotranslationally, during the biosynthesis of the protein and its
insertion into the membrane. Thus, although we propose a new topology,
it is possible that this specific region of Mdr is subjected to
reversible conformational changes in vivo, imposed by
substrates or other unknown effectors as recently suggested for the
effect of voltage on the open state conformation of a voltage-dependent
channel (Slatin et al., 1994). Mobility of membranous protein
segments is thought to involve unfavored energetic pathways. However,
assuming that these energetic problems could be accommodated for by
intra- or intermolecular interactions, we cannot rule out
``conformational adaptability'' (Theorell, 1967) in complex
membrane translocators.
, tetraphenylarsonium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.