(Received for publication, September 6, 1994)
From the
P-glycoprotein (Pgp) is a polytopic membrane protein responsible
for multidrug resistance in cancer cells. Previously, we have used a
coupled cell-free translation/translocation system to investigate the
membrane orientation of Pgp sequences and have made the unexpected
observation that predicted transmembrane (TM) segments from both the
NH-terminal and COOH-terminal halves inserted in microsomal
membranes in two different orientations (Zhang, J.-T., Duthie, M., and
Ling, V.(1993) J. Biol. Chem. 268, 15101-15110). How
these topological forms of Pgp are regulated is not known. In the
present study, we have used site-directed mutagenesis to investigate if
the amino acids surrounding the internal TM segments of Pgp may affect
their orientation. We discovered that the charged amino acids flanking
TM4 are important in determining the membrane orientation of the
NH
-terminal half molecule of Pgp. This is a novel
observation demonstrating the existence of internal topogenic sequences
in a mammalian polytopic membrane protein. These findings thus suggest
A) that the topological structure of a mammalian polytopic membrane
protein does not integrate into the membrane simply by following the
lead of the first inserted TM segment but that internal TMs may have
independent topogenic information and B) that the TM segments in a
multi-spanning membrane protein may be more dynamic than have been
previously anticipated, i.e. mutations in the amino acids
surrounding internal TMs could drastically change the overall topology
of the molecule.
P-glycoprotein (Pgp) ()is a polytopic membrane
protein responsible for multidrug resistance in cancer cells (1, 2, 3) . Pgp belongs to a superfamily of
ATP-binding cassette membrane protein transporters that includes the
cystic fibrosis transmembrane conductance regulator, peptide antigen
presentation-associated transporters, yeast STE6, and Escherichia
coli hemolysin transporter(4, 5, 6) .
Over 50 of these ATP-binding cassette transporters are known, and the
list of transported substrates is extremely diverse including inorganic
ions, amino acids, sugars, peptides, steroids, and hydrophobic
anticancer drugs. Substrate specificities of these transporters are
likely determined by their transmembrane domains. Hydropathy plot and
amino acid sequence analysis of Pgp suggested that it has 12
transmembrane (TM) segments and 2 ATP-binding
sites(7, 8, 9) . However, nascent Chinese
hamster pgp1 Pgp molecules synthesized in a cell-free
translation system in the presence of microsomal membranes (rough
microsomes) possess two different topological
structures(10, 11) . One has all 12 predicted TMs in
the membrane while the other has only 8 in the membrane. Similar
observations have been made with the COOH-terminal half of the molecule
of human MDR1 Pgp expressed in frog oocytes(12) .
Transmembrane proteins in eukaryotic cells are thought to acquire
their final membrane orientations during or immediately after synthesis
on the rough endoplasmic reticulum. Two theories have been proposed to
explain the orientation of membrane proteins. One is the
``positive-inside rule'' in which it is postulated that
membrane proteins orient themselves with the most positively charged
end in the cytoplasm(13, 14) . The second is the
``charge-difference rule'' in which membrane proteins orient
themselves according to the charge difference flanking the TM segment.
In a multi-spanning membrane protein, the charge difference flanking
the first (NH terminus) TM segment is thought to play a
dominant role in determining the overall membrane topology of the
protein. Subsequent TM segments are postulated to simply follow the
lead of the first one(15) . In this respect, the Pgp molecule
is unusual in that it occurs in more than one topological form. It has
been speculated that this feature of Pgp may be associated with its
multiple functions(11) . Pgp not only transports anticancer
drugs but may also function as an ATP channel (16) and a
cell-swelling activated chloride channel(17, 18) .
Gill et al.(19) have been able to separate
chloride-conducting function from drug-transport function of Pgp by
changing cell volume. These two functions may be generated by two
different conformations of Pgp.
In this study, we analyze how the two orientations of the hamster pgp1 Pgp observed in an in vitro translation system (10, 11) are regulated. Such in vitro systems have been used for studying in detail the topology of membrane proteins in a wide variety of systems(20) . These topological studies make use of physical and enzymatic properties of rough microsomes (RM) (derived from dog pancreatic endoplasmic reticulum) to detect the orientation and insertion of newly synthesized peptides. We have used site-directed mutagenesis to investigate the effects of charged amino acids on membrane orientation of Pgp sequences. Our results show that the charged amino acids surrounding the TM3 and TM4 determine their orientation in the membrane, independent of the first TM segment, TM1. This type of internal topogenic sequence may dictate the topologies of a wide range of polytopic transport proteins in mammalian cells.
Figure 1:
Coupled in vitro translation
with two membrane orientations of a truncated P-glycoprotein sequence. A, schematic linear structure of PGP-N4 molecule. The
full-length of PGP-N4 molecule consists of 550 amino acids, 4 TM
(TM1-TM4) segments (solidbars), and 4
consensus N-linked glycosylation sites
(&cjs1231;&cjs1231;). The arrow indicates the fusion
site between the NH
-terminal TM domain and the
COOH-terminal ATP-binding domain. Amino acids surrounding TM3 and TM4
are shown in single letter codes with charged amino acids marked by
(+) or(-). There are no charged amino acid residues within
the predicted TM segments. B, two observed membrane
orientations of PGP-N4 molecules in RM vesicles. In model I, both
NH
and COOH termini are on the cytoplasmic side (outside of
RM), and all four TM segments (solidbars) are in the
membrane. In model II, only three TM segments are in the membrane, and
the COOH terminus is in the RM lumen with an oligosaccharide chain
(&cjs1231;&cjs1231;
) attached. C, in vitro translation and endoglycosidase treatments of PGP-N4 molecules.
Two mature full-length products of 62 kDa (representing model II) and
59.5 kDa (representing model I) were translated from the in vitro transcripts in the presence of RM (lane 1). Lanes2-4 show limited endoglycosidase peptide N-glycosidase F (PNGaseF) treatment. The partially
deglycosylated peptide is indicated by arrows (lanes 2 and 3), and the fully deglycosylated peptide is 52 kDa (lane 4).
R207E, R207N, R207K/K210D, and R207V/K210D mutations in pGPGP-N3 were constructed from the corresponding pGPGP-N4 mutants. A EcoRI-BalI fragment containing the mutations were released from the pGPGP-N4 DNA and purified. A RsaI-HindIII fragment encoding the ATP-binding reporter was released from the wild type hamster pgp1 cDNA and purified. These two fragments were ligated together and cloned into a new pGEM-4z vector. The final DNA was sequenced to confirm the mutations and correct linkage between the two DNA fragments.
Previously, two orientations have been observed with the
NH-terminal half of the molecules of Chinese hamster pgp1 Pgp translated in vitro(11) . To
investigate determinants involved in the generation of the two
orientations of Pgp, we have made use of site-directed mutagenesis and
the pGPGP-N4 cDNA construct (11) to identify sequences
important for determining the topology of the NH
-terminal
half of Pgp. Fig. 1A shows the linear diagram of the
truncated Pgp molecule (named as PGP-N4) encoded by pGPGP-N4. The
PGP-N4 protein has been previously shown to be expressed in two
different orientations (Fig. 1B, see also (11) ). One orientation, representing about 25% of the
molecules (Fig. 1B, model I), has all four TMs
in the membrane whereas the other orientation, representing about 75%
of the molecules (Fig. 1B, model II), has only
three TM segments in the membrane, and the COOH terminus is located in
the RM lumen. The model I molecule has three N-linked
oligosaccharide chains in the extracellular loop linking TM1 and TM2,
while the model II molecule has an extra sugar chain in the
COOH-terminal tail (Fig. 1B). Thus, the model I
molecule has a faster mobility on SDS-polyacrylamide gel
electrophoresis than model II due to one less oligosaccharide chain
attached(11) .
Two major protein products (62 and 59.5 kDa)
were translated from the pGPGP-N4 RNA transcript in a rabbit
reticulocyte lysate supplemented with RM (Fig. 1C, lane 1; see also (11) ). The 62- and 59.5-kDa peptides
represent model II and model I molecules, respectively(11) .
Limited digestion of translation products with peptide N-glycosidase F generated intermediate deglycosylated products
attached with 1, 2, or 3 sugar chains (indicated by arrows in Fig. 1C, lanes 2 and 3). The fully
deglycosylated peptide is 52 kDa (Fig. 1C, lane 4). These results demonstrate that the 62-kDa peptide has
four oligosaccharide chains while the 59.5-kDa peptide has three.
Previously, we have shown that all of the COOH-terminal reporter
peptides protected from proteolysis have an oligosaccharide
chain(11) . Therefore, the possibility that the 59.5-kDa
peptide represents the model II molecules that do not have an
oligosaccharide chain in the COOH-terminal tail can be ruled out. The
proportion of the 62- and 59.5-kDa peptides serves as a convenient
indication for the relative amounts of model I and II molecules.
To
determine if the two positively charged amino acids (Arg-207 and
Lys-210) between TM3 and TM4 (Fig. 1A) have topogenic
information for the membrane orientation of the PGP-N4 molecule, we
made mutations of these two amino acids using site-directed mutagenesis
and generated PGP-N4-R207E, -R207N, -R207K/K210D, and -R207V/K210D
mutant cDNAs (Fig. 2A). Fig. 2B shows
the cell-free translation product from wild type (WT) and mutant
templates. About 25% of membrane-associated full-length products of WT
have model I (59.5 kDa) orientation, and the remaining 75% have model
II (62 kDa) orientation (Fig. 2B, lane 1; see
also Table 1). The R207E and R207N mutants generated 70%
model I and
30% model II molecules (Fig. 2B, lanes 2 and 3). The double mutant R207K/K210D
generated 85% model I and 15% model II molecules (Fig. 2B, lane 4). Essentially no model II
molecules were generated from the double mutant R207V/K210D (Fig. 2B, lane 5). These observations were
confirmed by proteolysis/membrane protection assays of translation
products. Upon protease K treatment, the WT, R207N, R207E, and
R207K/K210D generated a
42-kDa protease-resistant and glycosylated
fragment (COOH-terminal tail located in RM lumen), whereas the
R207V/K210D did not (data not shown). Limited peptide N-glycosidase F treatment shows that the major product from
R207K/K210D (Fig. 2D, lanes 1-4) and
R207V/K210D (Fig. 2D, lanes 5-8) mutants
has three oligosaccharide chains (Fig. 1B, model
I). These results indicate that both the Arg-207 and Lys-210
between TM3 and TM4 affect the membrane orientation of PGP-N4
molecules. It has been previously suggested that Arg residues have more
restrictive effects than Lys residues(21) . This may explain
why fewer model II molecules were generated from R207K/K210D than from
R207E, although both have similar charge changes. R207E and R207N
mutations have similar effects on the membrane topology of PGP-N4,
suggesting that the addition of a negative charge does not
significantly affect the membrane topology. The origin of the minor
band below the major product of R207K/K210D mutant (lane 4) is
not known, and it is not consistently observed (see Fig. 2D). Its presence does not affect our
interpretation of the experimental data. It should be noted that the
mobility difference observed with mutant PGP-N4-R207K/K210D and
PGP-N4-R207V/K210D is likely due to charge changes (Fig. 2B, lanes 4 and 5). The effect
of charges on the mobility of truncated Pgp peptides has also been
previously observed(22) . Sequencing the mutant PGP-N4 cDNA
shows no mutation other than the designed ones. When the translation
was per-formed in the absence of RM, the mobility of mutant precursors
is also different from the WT (Fig. 2C). This shows
that the difference in mobility is not due to modification caused by
RM-associated enzymes.
Figure 2:
Membrane orientation of PGP-N4 molecules
with mutations NH-terminal to TM4. A, amino acid
sequences flanking TM4 of WT and mutant PGP-N4 molecules. The 7 amino
acids at NH
terminus and 15 amino acids at COOH terminus of
TM4 (hatchedbar) are shown. The dashedlines denote sequence identity of mutants with the WT
molecule. The mutations at NH
terminus to TM4 are shown at
specific positions. Net charge of NH
-terminal (N)
and COOH-terminal (C) sequences of TM4 and the total net
charge (
(C-N)) flanking TM4 are shown on the left with the name of each construct. B and C,
expression of WT and mutant PGP-N4 molecules. WT and mutant PGP-N4
molecules were translated in the presence (panel B) or absence (panel C) of RM. The membrane fraction (panel B) or
total fraction in the absence of RM (panel C) was analyzed by
SDS-polyacrylamide gel electrophoresis. I and II in lane 1 of panel B denote molecules with model I and
II orientations shown in Fig. 1B. D, limited
endoglycosidase treatment of R207K/K210D and R207V/K210D mutant
molecules. The membrane-associated translation products were treated
with 0 (lanes 1 and 5), 13 (lanes 2 and 6), 17 (lanes 3 and 7), and 60 milliunits (lanes 4 and 8) of peptide N-glycosidase F. Arrows denote the intermediate
products.
The fact that a reduction in positive charge
in the domain linking TM3 and TM4 generates more model I molecules
suggests that these positive charges function as a restriction factor
causing TM4 to insert into the membrane with its amino terminus in
cytoplasm. This is consistent with previous studies on mammalian type
II membrane proteins (23) and asialoglycoprotein receptor H1 (24) where the positive charges at the NH terminus
of the TM segment are retained in the cytoplasmic side. In both
instances, a membrane protein with a single TM segment was used. The
current study is the first to demonstrate that charged amino acids
flanking an internal (or subsequent) TM segment of a mammalian
polytopic membrane protein are important in determining the membrane
topology.
To investigate if the charged amino acids at the COOH terminus of TM4 also affect the membrane orientation of PGP-N4 molecules, we constructed PGP-N4-D238K, -D238K/E240R, and -K231E mutant molecules (Fig. 3A). The translation results of these mutant molecules are shown in Fig. 3B. It can be seen that while the WT molecule has about 75% model II orientation, the D238K and D238K/E240R mutants have 59 and 36% model II, respectively (Fig. 3B, Table 1). This suggests that adding positive charges in the COOH terminus of TM4 generates less model II molecules. The mutation at Lys-231 (mutant K231E) also appears to have increased slightly the proportion of model II molecules. These results, together with that in Fig. 2, indicate that the more positively charged domain tends to be retained in the cytoplasmic side of the membrane.
Figure 3:
Membrane orientation of PGP-N4 molecules
with mutations COOH-terminal to TM4. A, amino acid sequences
flanking TM4 of WT and mutant PGP-N4 molecules. The 7 amino acids at
NH terminus and 15 amino acids at COOH terminus to TM4 (hatchedbar) are shown. The dashedlines denote sequence identity of mutants with the WT
molecule. The mutations at COOH terminus to TM4 are shown at specific
positions. Net charge of NH
terminus (N) and COOH
terminus (C) sequences of TM4 and the total net charge (
(C-N)) flanking TM4 are shown on the left. B, expression of WT and PGP-N4 molecules. I and II in lane 1 denote molecules with model I and II
orientations.
The nature and position of the charged amino acids may
also play some role in determining membrane orientation. For example, a
similar charge change across TM4 appears to generate different effects
on the orientation of TM4. As shown in Fig. 2and Fig. 3,
the (N-C) (total net charge of NH
- and COOH-terminal
sequences of TM4) of both R207E and D238K mutants equals zero; however,
these two mutations yielded molecules with different ratios between the
two orientations (Table 1). Moreover, mutations at the NH
terminus appear to affect the topology of PGP-N4 molecules more
than mutations at the COOH terminus of TM4. This is probably due to the
fact that the charges at the COOH terminus are farther away from TM4
and therefore have less effect than the ones at the NH
terminus(25) . Alternatively, the amino acids at the
NH
terminus of TM4 affect TM3 as well as TM4, while the
charged amino acids at the COOH terminus affect only TM4.
To study whether Arg-207 and Lys-210 affect the membrane orientation of TM3, we engineered mutations of Arg-207 and Lys-210 into PGP-N3 molecule, which has only TM1, TM2, and TM3 with an ATP-binding COOH-terminal tail (Fig. 4A). The wild type PGP-N3 is expressed as 40% model I and 60% model II molecules (Fig. 4B; see also Fig. 4C, lane 1, and Table 1). When only one positive charge (Arg-207 or Lys-210) was changed to neutral or negative charge, model I molecules were increased slightly compared with WT (Fig. 4C, lanes 2, 3, and 5; see also Table 1). When both positive charges were changed to neutral and negative charges (R207V/K210D), all of the molecules had the model I orientation. These results suggest that Arg-207 and Lys-210 also affect the membrane orientation of TM3 but to a lesser extent than TM4.
Figure 4:
Membrane insertion and orientation of
PGP-N3 molecules with mutations COOH-terminal to TM3 A,
schematic linear structure of PGP-N3 molecule. The full length of
PGP-N3 molecule consists of three predicted transmembrane segments (solidbars) and four consensus N-linked
glycosylation sites (&cjs1231;&cjs1231;). The arrow indicates the fusion site between the NH
-terminal TM
domain and the COOH-terminal ATP-binding domain. Amino acids
surrounding the TM3 are shown in single letter code with charged amino
acids marked by (+) or(-). B, two models of
membrane orientations of PGP-N3 molecules in RM vesicles. The model I
molecule has all three TM segments (solidbars) in
membrane with NH
and COOH termini on the different sides of
RM membranes. An extra oligosaccharide chain is attached to the
COOH-terminal domain of the model I molecule. Both NH
and
COOH termini of the model II molecule are in cytoplasmic side (outside
of RM) with the TM3 located on the outside of RM. C,
expression of WT and mutant PGP-N3 molecules. I and II in lane 1 denote molecules with model I and II
orientations. D, amino acid sequences flanking TM3 of WT and
mutant PGP-N3 molecules. The 15 amino acids on both sides flanking TM3 (hatchedbar) are shown. The dashedlines denote sequence identity of mutants with the WT.
The mutations are shown at specific positions on the COOH terminus of
TM3. Net charge of NH
-terminal (N) and
COOH-terminal (C) sequences of TM3 and the total net charge (
(C-N)) flanking TM3 are shown on the left.
Using Chinese hamster pgp1 Pgp sequences as a model
system, we showed that internal TM segments of a mammalian polytopic
membrane protein have topogenic information. We showed that the charged
amino acids flanking the TM3 and TM4 of hamster pgp1 Pgp are
very important in determining their relative membrane orientation in
the in vitro translation system. Deletion of positive charges
at the NH-terminal side or addition of positive charges at
the COOH-terminal side of the TM4 segment have similar effects on the
membrane orientation of TM4. In both cases, more model I molecules were
generated. This observation is unique and has not been previously
demonstrated for other mammalian polytopic membrane proteins.
Hartmann et al.(15) postulated that TM segments in
a polytopic membrane protein simply follow the lead of the first
inserted TM segment and insert sequentially into the membrane. This is
supported by the observation that the membrane insertion of the
subsequent TMs in an artificial polytopic membrane protein does not
depend on the signal-recognition particle and the signal recognition
particle receptor(26) . Our current study, however, showed that
the sidedness of membrane insertion of the TM3 and TM4 segments of Pgp
can follow a different pathway. The orientation of TM3 and TM4 in the
membrane is dependent on their surrounding charges and does not simply
follow the lead of the first TM segment. We hypothesize that the
presence of positive charges at the NH terminus of TM4
creates a cytoplasmic ``retention signal'' for the NH
terminus of TM4, and thus, model II molecules of PGP-N4 are
generated (see Fig. 1). The removal of this positive charge
retention signal by site-directed mutagenesis defaults PGP-N4 to a
model I probability. Lack of positive charges at the COOH terminus of
TM4 may also decrease the potential for TM4 insertion into the membrane
in model I orientation. These findings are not in agreement with the
prevailing idea that TM segments in a polytopic membrane protein follow
the lead of the first inserted TM and insert sequentially. Internal TM
segments may have their own topogenic sequences, and mutations of these
amino acids may drastically change their insertion and orientation in
membrane. This type of internal topogenic sequence may dictate
topologies of a wide range of polytopic transport proteins in mammalian
cells.
Although it remains to be demonstrated, our hypothesis that more than one topological structure of Pgp is expressed in mammalian cells has functional implications. It is possible that these different topological structures of Pgp are associated with its multiple functions(11, 12) . Pgp not only transports anticancer drugs but also functions as an ATP channel (16) and a cell-swelling activated chloride channel(17, 18, 19) . Gill et al.(19) have been able to separate two conformations of Pgp that are responsible for the chloride channel and drug transport functions. Further studies with site-directed mutagenesis and transfection into mammalian cells to evaluate function should provide insight into the validity of the above hypothesis.