Báo cáo khoa học: Functional role of the linker region in purified human P-glycoprotein pot

Functional role of the linker region in purified human
P-glycoprotein
Tomomi Sato
1
, Atsushi Kodan
2
, Yasuhisa Kimura
3
, Kazumitsu Ueda
2,3
, Toru Nakatsu
1
and
Hiroaki Kato
1
1 Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
2 Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan
3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Human P-glycoprotein (P-gp, ABCB1), which conveys
multidrug resistance, is a drug efflux pump that trans-
ports a wide variety of structurally unrelated com-
pounds out of cells [1–4]. The transport by P-gp is
driven by energy from ATP hydrolysis, and P-gp is
classified as a member of the ATP-binding cassette
(ABC) transporter family [5,6].
The transport of substrate by P-gp is thought to be
coupled with ATP hydrolysis [7]. Without a transport
substrate, P-gp has low basal ATP hydrolase (ATPase)
activity, whereas with substrates P-gp exhibits high
ATPase activity, which is known as substrate-stimu-
lated ATPase activity [8–11]. Thus, the substrate-stim-

ulated ATPase activity can be a measure of the
recognition of substrate by P-gp. When titrating P-gp
substrates, the activity increases up to a maximum but
then decreases at high substrate concentrations. This
characteristic bell-shaped activity profile has been
Keywords
ATPase activity; limited proteolysis; linker
region; MDR1; P-glycoprotein
Correspondence
H. Kato, Graduate School of Pharmaceutical
Sciences, Kyoto University, 46-29
Yoshida-Shimo-Adachi-cho, Sakyo-ku,
Kyoto 606-8501, Japan
Fax: +81 75 753 9272
Tel: +81 75 753 4617
E-mail:
(Received 28 December 2008, revised 19
April 2009, accepted 23 April 2009)
doi:10.1111/j.1742-4658.2009.07072.x
Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an
ATP-dependent drug efflux pump that transports a wide variety of struc-
turally unrelated compounds out of cells. P-gp possesses a ‘linker region’
of $ 75 amino acids that connects two homologous halves, each of which
contain a transmembrane domain followed by a nucleotide-binding
domain. To investigate the role of the linker region, purified human P-gp
was cleaved by proteases at the linker region and then compared with
native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-
exclusion chromatography analysis and a thermo-stability assay, cleavage
of the P-gp linker did not directly affect the preservation of the overall
structure or the catalytic process in ATP hydrolysis. However, linker cleav-

age increased the k
cat
values both with substrate (k
sub
) and without
substrate (k
basal
), but decreased the k
sub
⁄ k
basal
values of all 10 tested
substrates. The former result indicates that cleaving the linker activates
P-gp, while the latter result suggests that the linker region maintains the
tightness of coupling between the ATP hydrolase reaction and substrate
recognition. Inspection of structures of the P-gp homolog, MsbA, suggests
that linker-cleaved P-gp has increased ATP hydrolase activity because the
linker interferes with a conformational change that accompanies the ATP
hydrolase reaction. Moreover, linker cleavage affected the specificity con-
stants [k
sub
⁄ K
m(D)
] for some substrates (i.e. linker cleavage probably shifts
the substrate specificity profile of P-gp). Thus, this result also suggests that
the linker region regulates the inherent substrate specificity of P-gp.
Abbreviations
ABC, ATP-binding cassette; ATPase, ATP hydrolase; calcein-AM, 3¢,6¢-di(O-acetyl)-4¢,5¢-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein
tetraacetoxymethyl ester; NBD, nucleotide-binding domain; P-gp, P-glycoprotein; PIPES, piperazine-N,N’-bis(2-ethanesulfonic acid); TM,
transmembrane; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; b-UDM, n-undecyl-b-

D-maltopyranoside.
3504 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS
analyzed using modified Michaelis–Menten kinetics
[12–15]. The kinetic models used to evaluate bell-
shaped activity curves take into account an activating
substrate-binding step at low substrate concentrations
and an inhibitory drug-binding step at high substrate
concentrations, followed by a catalytic ATP-hydrolysis
step.
P-gp is a 1280-amino acid polypeptide divided into
two highly homologous halves: the N-half and the
C-half [16]. Each half contains a transmembrane (TM)
domain consisting of six TM helices followed by a
nucleotide-binding domain (NBD) [16]. These two
halves are connected by a ‘linker region’ of $ 75
amino acids that spans the region from approximately
Glu633 to Tyr709 [16–18].
It is fascinating and still unknown why the two
halves of P-gp are connected by a linker region, while
the two halves of bacterial [19] and some mammalian
ABC transporters, such as ABCG family members
[20], are not connected by a linker region and act as
homodimers or heterodimers. Recently, limited prote-
olysis of the linker region has shed light on its involve-
ment in the ATPase activity of P-gp. The linker region
has been shown to be highly susceptible to different
proteases [21–24], and the trypsin and chymotrypsin
cleavage sites were identified as Arg680 and Leu682,
respectively [22]. Trypsin cleavage increased the basal,
verapamil-stimulated and vinblastine-stimulated

ATPase activities, suggesting that cleavage of the
linker activates P-gp [21]. By contrast, cleavage with
chymotrypsin or proteinase K decreased the verapa-
mil-stimulated and vinblastine-stimulated ATPase
activities, even though the basal and colchicine-stimu-
lated ATPase activities increased upon cleavage [22].
This disproportional alteration between basal and
substrate-stimulated ATPase activity upon cleavage
suggests that ATP hydrolysis and transport are proba-
bly uncoupled by cleavage of the linker [22]. Although
these results provided valuable suggestions for the role
of the P-gp linker region, the molecular details, such
as the involvement of the linker region in substrate
recognition, are still unclear. In addition, it is still
unknown why different proteases caused various
changes in the ATPase activity of P-gp. As all previous
studies used crude membrane fractions containing
P-gp, the results could be affected by other proteins.
In fact, it was reported that the linker region of P-gp
interacts with other proteins such as RING finger
protein 2 (RNF2) [25] and both alpha-tubulin and
beta-tubulin [26]. Therefore, in order to investigate
how the ATPase activity of P-gp is modulated by alter-
ations in the linker region, it would be preferable to
use a highly purified preparation of P-gp. Purified
P-gp will not only exclude the effects of interacting
proteins but can also be used to perform kinetic analy-
ses of the native and linker-cleaved P-gp.
In the present study, we investigated the functional
role of the linker region of human P-gp using a highly

purified and properly folded protein preparation [27].
We performed limited proteolysis experiments on P-gp
and confirmed that the linker region was the site most
susceptible to protease digestion; we also identified five
cleavage sites, four of which were novel. Using a P-gp
preparation in which the linker had been cleaved by
trypsin and chymotrypsin, we measured the basal and
substrate-stimulated ATPase activities for 10 transport
substrates. This kinetic analysis provided new insight
into the role of the linker region. In addition, we fur-
ther analyzed the functional role of the linker region
based on the crystal structures of a P-gp homolog,
MsbA [28].
Results
Protease treatment of purified P-gp in detergent
micelles
To investigate the role of the linker region of P-gp, a
linker-cleaved P-gp was generated by protease cleavage
because the linker region is highly susceptible to prote-
ase cleavage [21–24]. Highly purified P-gp in detergent
micelles was incubated with trypsin, chymotrypsin, V8
protease, or subtilisin, as indicated in the Materials
and methods. Despite the different substrate specifici-
ties of these proteases, all cleaved P-gp in a similar
pattern and generated two fragments, with molecular
masses of 67–69 and 60–65 kDa, as determined using
SDS–PAGE (Fig. 1).
Trypsin cleavage kinetics of reconstituted P-gp
and its residual ATPase activity
The relationship between the degree of trypsin cleav-

age of P-gp and its residual verapamil-stimulated
ATPase activity was investigated. Reconstituted P-gp
was readily cleaved into two fragments, of 69 and
60 kDa, by SDS–PAGE (Fig. 2A). Further cleavage
generated a 56 kDa fragment. As P-gp cleavage pro-
ceeded, the verapamil-stimulated ATPase activity
gradually increased to a maximum of 340% within
105 min. The increase in verapamil-stimulated
ATPase activity correlated with a decrease in the
residual amount of native P-gp (Fig. 2B). This
increase in verapamil-stimulated ATPase activity was
also observed following cleavage with chymotrypsin,
as described below.
T. Sato et al. Functional role of the linker region in P-gp
FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3505
Identification of the cleavage sites by N-terminal
amino acid sequence analysis
To determine the protease cleavage sites, we performed
N-terminal amino acid sequence analysis by Edman
degradation. The results are summarized in Table 1,
and the corresponding fragments on SDS–PAGE are
shown in Figs 1 and 2A and in Fig. S1 as (A–E).
These data identified fragments A, B, C and D as the
C-terminal side fragments from Arg680, Leu662,
Glu652 and Lys685, respectively. All of these cleavage
sites are located in the P-gp linker region from Glu633
to Tyr709. Thus, the two fragments shown in Fig. 1
occurred upon cleavage of the linker region and were
thereby identified as the N-half and C-half fragments
of P-gp. Fragment E had a molecular mass of 37 kDa

and was also generated by extensive trypsin digestion
(shown in Fig. S1). The N-terminal amino acid
sequence analysis identified fragment E as the C-termi-
nal fragment from Arg933, which was predicted to be
located on the cytoplasmic side of the TM 11 helix in
a Sav1866-based homology model of P-gp [29].
Size-exclusion chromatography analysis
To investigate the structural properties of trypsin-
cleaved P-gp, we performed size-exclusion chromato-
graphy. As shown by the arrows in lane 2 of Fig. 3A,
all P-gp molecules were cleaved by trypsin into two
fragments that migrated at 60 and 69 kDa when ana-
lyzed using SDS–PAGE. The trypsin-cleaved P-gp and
the native P-gp were subjected to size-exclusion chro-
matography (Fig. 3B). The trypsin-cleaved P-gp eluted
as a sharp peak at the same retention volume as the
native P-gp (Fig. 3B). To further corroborate this
result, the peak fractions of trypsin-cleaved P-gp were
collected and rechromatographed, resulting in elution
at the same retention volume as that of native P-gp
(data not shown). These results strongly indicate that
the N-half and C-half fragments of P-gp retain a stable
interaction, even after the linker is cleaved.
A
B
Fig. 2. Trypsin treatment and residual ATPase activity of reconsti-
tuted P-gp. (A) Time course of trypsin treatment analyzed using
SDS–PAGE followed by silver staining. (B) The verapamil-stimulated
ATPase activity of P-gp with (d) or without (
) trypsin treatment,

and the remaining amount of P-gp (s). The verapamil-stimulated
ATPase activity before protease digestion was set to a value of
100%. The remaining amounts of native P-gp (%) were quantified
using
IMAGE J software (National Institutes of Health). In ATPase
activity measurement, all data points represent the means ± SD
from three independent assays. Error bars are shown unless they
are smaller than the symbol.
Fig. 1. SDS–PAGE of P-gp cleaved by various proteases. The SDS–
PAGE lanes are as follows: lane 1, native P-gp; lane 2, trypsin-
cleaved P-gp (5-min incubation); lane 3, chymotrypsin-cleaved P-gp
(30-min incubation); lane 4, V8 protease-cleaved P-gp (15-min
incubation); lane 5, subtilisin-cleaved P-gp (5-min incubation). The
fragment to the right of each asterisk was identified by N-terminal
amino acid sequence analysis.
Functional role of the linker region in P-gp T. Sato et al.
3506 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS
Comparison of the thermostability of native and
trypsin-cleaved P-gp
To examine the effect of protease cleavage on the thermo-
stability of P-gp, the residual ATPase activities of
native and trypsin-cleaved P-gp were examined after
incubation at various temperatures. Reconstituted P-gp
(0.01 mgÆmL
)1
) was treated with trypsin at a P-gp :
trypsin ratio of 40 : 1 (w ⁄ w) at 20 °C for 3 h, which
resulted in no residual native P-gp. After incubation
of the native P-gp and the trypsin-cleaved P-gp at 40, 45,
or 50 °C, the verapamil-stimulated ATPase activities

were measured. As shown in Fig. 4, the residual ATPase
activities of trypsin-cleaved P-gp and of native P-gp were
similar after the heat treatments. This result indicates
that there is no effect of trypsin cleavage on the thermo-
stability of P-gp.
Comparison of MgATP affinity between the
native P-gp and the trypsin-cleaved P-gp
To examine the effect of trypsin cleavage on the affin-
ity for MgATP, we compared the K
m
values for
MgATP, designated K
m(MgATP)
. The reconstituted P-gp
was treated with trypsin at a P-gp : trypsin ratio of
200 : 1 (w ⁄ w), at 20 °C for 1.5 h. The ATPase activi-
ties were measured in the presence of 50 lm verapamil
and various concentrations of MgATP. There were no
considerable differences in the K
m(MgATP)
values
between the native P-gp and the trypsin-cleaved P-gp,
which were 0.59 ± 0.33 and 0.89 ± 0.37 mm, respec-
tively. Thus, the linker-cleaved P-gp and the native
P-gp have similar affinities for MgATP.
Kinetic properties of the native P-gp and the
protease-cleaved P-gp with respect to several
transport substrates
To investigate the effect of linker cleavage on the
recognition of various transport substrates, we deter-

mined the kinetic parameters of P-gp ATPase activity
with respect to 10 transport substrates. The chemical
structures of the transport substrates tested in this
study are shown in Fig. S2.
Figure 5A shows the initial rates of ATP hydrolysis
as a function of the verapamil concentration. The
Table 1. Results of N-terminal amino acid sequence analysis.
Corresponding
fragments
a
Cleavage
conditions
b
Protease
N-terminal
sequence obtained
Sequence surrounding
cleavage site
c
Position within
structure
A I Trypsin KLSTK
677
AQDRflKLSTK Linker region
B I Chymotrypsin IRKRS
659
RSSLflIRKRS Linker region
C I V8 protease M(S ⁄ L)XM(D ⁄ E)
649
DALEflMSSND Linker region

D II Trypsin EALDE
682
LSTKflEALDE Linker region
E II Trypsin KAHIF
930
NSLRflKAHIF Membrane surface
d
a
Fragment A is shown in Figs 1 and 2A, fragments B and C are shown in Fig. 1, fragment D is shown in Figs 2A and S1, and fragment E is
shown in Fig. S1.
b
Detailed cleavage conditions are described in the Materials and methods.
c
The identified cleavage sites are denoted by
arrows (fl).
d
The cleavage site of fragment E was located on the cytoplasmic side of the TM 11 helix.
A
B
Fig. 3. Size-exclusion chromatography.
Purified P-gp was incubated with trypsin
at a P-gp : trypsin ratio of 200 : 1 (w ⁄ w)
at 20 °C for 30 min. The cleaved P-gp
was analyzed using SDS–PAGE and size-
exclusion chromatography. (A) SDS–PAGE
of injected samples. Lane 1, native P-gp;
lane 2, trypsin-treated P-gp. (B) Size-
exclusion chromatography profiles of native
(dotted line) and trypsin-treated (solid line)
P-gp.

T. Sato et al. Functional role of the linker region in P-gp
FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3507
ATPase profiles of trypsin-cleaved P-gp and of chymo-
trypsin-cleaved P-gp showed a characteristic pattern
[30,31], similar to that of native P-gp. ATPase activity
was stimulated in the presence of low substrate concen-
trations but was inhibited in the presence of high
substrate concentrations. These three curves were fitted
by the modified Michaelis–Menten equation (Eqn 1)
[13], and kinetic parameters were calculated as listed in
Table 2. Similar curves were obtained with rhodamine
123, colchicine, nicardipine, rhodamine B and vinblas-
tine, and the curves were fitted by Eqn (1) (data not
shown). With the other substrates [nicardipine, digoxin,
paclitaxel, 6¢-di(O-acetyl)-4¢,5¢-bis[N,N-bis(carboxym-
ethyl)aminomethyl]fluorescein tetraacetoxymethyl ester
(calcein-AM) and valinomycin], the simple Michaelis–
Menten equation (Eqn 2) was used to calculate kinetic
parameters because the curves did not obey Eqn (1)
(Fig. 5B).
The kinetic parameters for various structurally
unrelated transport substrates obtained with native,
trypsin-cleaved and chymotrypsin-cleaved P-gp are
summarized in Table 2. The k
basal
values obtained with
native, trypsin-cleaved and chymotrypsin-cleaved P-gp
were 0.189, 2.38 and 2.95 s
)1
, respectively. Thus, linker

cleavage increased the k
basal
value, as reported
previously [21,22]. Likewise, for each substrate, the k
sub
values obtained with protease-cleaved P-gp were higher
than those obtained with native P-gp. Thus, linker
cleavage also increased the k
sub
value. This result is
inconsistent with a previous report that chymotrypsin
cleavage decreased the verapamil-stimulated and
vinblastine-stimulated ATPase activities [21,22].
However, the crude membrane used in these previous
Residual ATPase activity (%)
0
20
40
60
80
100
0 5 10 15 20
Time of incubation (min)
40 °C
45 °C
50 °C
Trypsin-cleaved P-gp
Native P-gp
Fig. 4. Residual ATPase activity after heating at 40, 45 and 50 °C.
The residual ATPase activity profile for native P-gp incubated at

40 °C(
), 45 °C( ) and 50 °C(d), and that for trypsin-cleaved
P-gp incubated at 40 °C(h), 45 °C(4) and 50 °C(s), are shown.
The ATPase activity was measured in the presence of 50 l
M verap-
amil. All data points represent the means ± SD from three indepen-
dent assays. Error bars are shown unless they are smaller than the
symbol.
0
6
4
2
8
1
Verapamil (µ
M)
ATPase activity (s
–1
)
9
10
–1
10
1
10
2
10
3
10
4

1
3
5
7
Native
Trypsin
Chymotrypsin
10
A B
ATPase activity (s
–1
)
Valinomycin (µM)
0
1
2
3
4
5
6
0
4
2
1
3
5
6
7
8
9

10
11
12
Native
Trypsin
Chymotrypsin
Fig. 5. Substrate concentration dependence of native and protease-cleaved P-gp ATPase activity. Purified and reconstituted P-gp was incu-
bated with trypsin, chymotrypsin, or no protease at 20 °C for 1.5 h. The ratio of P-gp : trypsin and P-gp : chymotrypsin was 200 : 1 (w ⁄ w)
and 100 : 1 (w ⁄ w), respectively. The ATPase activities of native (s), trypsin-cleaved (
) and chymotrypsin-cleaved ( ) P-gp were measured
in the presence of various concentrations of transport substrates. All data points represent the means ± SD from three independent assays.
Error bars are shown unless they are smaller than the symbol. (A) The ATPase profiles with various concentrations of verapamil. Solid lines
are fits to the modified Michaelis–Menten equation (Eqn. 1). (B) The ATPase profiles with various concentrations of valinomycin. Solid lines
are fits to the simple Michaelis–Menten equation (Eqn. 2)
Functional role of the linker region in P-gp T. Sato et al.
3508 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS
studies may have contained contaminating proteins that
interact with P-gp [25,26] and cause this inconsistency.
The k
sub
values ranged from 0.410 to 3.48 s
)1
, while the
k
basal
value was 0.189 s
)1
for native P-gp. Thus,
substrate-stimulated ATPase activities were clearly
observed for native P-gp, as previously described

[8–10,31]. For trypsin-cleaved P-gp and chymotrypsin-
cleaved P-gp, the k
sub
values ranged from 3.44 to
11.1 s
)1
and from 3.69 to 11.7 s
)1
, while the k
basal
values
were 2.38 and 2.95 s
)1
, respectively. Thus, substrate-
stimulated ATPase activities were also observed for
protease-cleaved P-gp. The fold stimulation of the
ATPase activity for each substrate is represented as
k
sub
⁄ k
basal
values. For each substrate, the k
sub
⁄ k
basal
Table 2. Kinetic parameters of native and protease-cleaved P-gp. Reconstituted P-gp (0.01–0.03 mgÆmL
)1
) was incubated with either trypsin
or chymotrypsin at 20 °C for 1.5 h. The P-gp : trypsin and P-gp : chymotrypsin ratios were 200 : 1 and 100 : 1, respectively. Native P-gp
was prepared by incubating 0.01–0.03 mgÆmL

)1
of reconstituted protein without proteases at 20 °C for 1.5 h. The cleavage was stopped
and ATPase activity was measured as indicated in the Materials and methods. All data represent the means ± SD from three independent
assays.
Transport substrates
a
Protease
treatment k
basal
b
(s
)1
) k
sub
c
(s
)1
)
k
sub
⁄ k
basal
d
(-fold) K
m(D)
(lM)
k
sub
⁄ K
m(D)

(s
)1
ÆmM
)1
)
No substrate Native 0.189 ± 0.093
Trypsin 2.38 ± 0.33
Chymotrypsin 2.95 ± 0.41
Rhodamine 123 (342) Native 1.84 ± 0.01 9.8 ± 4.8 35.4 ± 1.2 52.0 ± 1.8
Trypsin 8.19 ± 0.24 3.4 ± 0.5 11.8 ± 3.5 692 ± 203
Chymotrypsin 8.56 ± 0.25 2.9 ± 0.4 17.5 ± 5.7 489 ± 158
Colchicine (399) Native 1.25 ± 0.03 6.2 ± 3.1 720 ± 48 1.66 ± 0.12
Trypsin 7.81 ± 0.02 2.9 ± 0.5 278 ± 24 28.2 ± 2.4
Chymotrypsin 6.40 ± 0.21 2.5 ± 0.5 280 ± 72 22.9 ± 5.9
Verapamil (440) Native 3.28 ± 0.13 17 ± 9 4.23 ± 0.84 775 ± 156
Trypsin 8.27 ± 0.12 3.5 ± 0.5 0.614 ± 0.139 13 500 ± 3100
Chymotrypsin 7.71 ± 0.18 2.6 ± 0.4 0.637 ± 0.276 12 100 ± 5300
Nicardipine (479) Native 3.48 ± 0.14 18 ± 9 1.91 ± 0.21 1820 ± 210
Trypsin 8.44 ± 0.46 3.6 ± 0.5 0.652 ± 0.442 12 900 ± 8800
Chymotrypsin 7.80 ± 0.30 2.6 ± 0.4 0.383 ± 0.151 20 400 ± 8100
Rhodamine B (479) Native 2.23 ± 0.07 12 ± 6 29.8 ± 6.0 74.9 ± 15.1
Trypsin 6.99 ± 0.33 2.9 ± 0.4 10.9 ± 1.2 641 ± 76
Chymotrypsin 7.22 ± 0.26 2.5 ± 0.4 14.2 ± 1.2 509 ± 47
Digoxin (781) Native 0.791 ± 0.033 4.2 ± 2.1 187 ± 16 4.23 ± 0.41
Trypsin 5.65 ± 0.08 2.4 ± 0.3 119 ± 21 47.3 ± 8.3
Chymotrypsin 5.99 ± 0.07 2.0 ± 0.3 101 ± 16 59.2 ± 9.5
Vinblastine (808) Native 1.90 ± 0.07 10 ± 5 1.21 ± 0.12 1560 ± 168
Trypsin 6.39 ± 0.31 2.7 ± 0.4 2.01 ± 0.23 3170 ± 389
Chymotrypsin 6.54 ± 0.23 2.2 ± 0.3 2.36 ± 0.92 2770 ± 1090
Paclitaxel (849) Native 0.410 ± 0.017 2.2 ± 1.1 0.874 ± 0.039 469 ± 29

Trypsin 3.44 ± 0.11 1.5 ± 0.2 0.775 ± 0.232 4440 ± 1340
Chymotrypsin 3.69 ± 0.05 1.3 ± 0.2 0.665 ± 0.272 5630 ± 2340
Calcein-AM (996) Native 3.19 ± 0.05 17 ± 8 2.03 ± 0.70 1570 ± 550
Trypsin 11.2 ± 0.7 4.7 ± 0.7 2.43 ± 0.41 4590 ± 830
Chymotrypsin 11.2 ± 0.2 3.8 ± 0.5 3.35 ± 0.37 3360 ± 380
Valinomycin (1,111) Native 2.95 ± 0.17 16 ± 8 0.395 ± 0.082 7460 ± 1610
Trypsin 11.1 ± 0.3 4.7 ± 0.7 1.11 ± 0.25 10 000 ± 2300
Chymotrypsin 11.7 ± 0.5 4.0 ± 0.6 1.51 ± 0.48 7770 ± 2470
a
Values in parentheses indicate the relative molecular mass of each transport substrate.
b
k
basal
is the k
cat
value for basal ATPase activity
with no transport substrates.
c
k
sub
is the k
cat
value for substrate-stimulated ATPase activity with each transport substrate.
d
k
sub
⁄ k
basal
represents the fold stimulation by each transport substrate.
T. Sato et al. Functional role of the linker region in P-gp

FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3509
values of trypsin-cleaved P-gp and chymotrypsin-
cleaved P-gp were lower than those of native P-gp. Thus,
the fold stimulation by the transport substrate decreased
with linker cleavage. There were some differences in the
K
m(D)
values between the protease-cleaved P-gp and
native P-gp. With digoxin, vinblastine, paclitaxel and
calcein-AM, the K
m(D)
values for each substrate
obtained with protease-cleaved P-gp were similar to
those obtained with the native P-gp, and the differences
in the K
m(D)
values between protease-cleaved P-gp and
native P-gp were within twofold. With rhodamine 123,
colchicine, verapamil, nicardipine and rhodamine B, the
K
m(D)
values obtained with protease-cleaved P-gp were
two to sevenfold lower than those obtained with native
P-gp. With valinomycin, the K
m(D)
values obtained with
protease-cleaved P-gp were three to fourfold higher than
those obtained with native P-gp. The k
sub
⁄ K

m(D)
values
obtained with protease-cleaved P-gp were 1 to 17-fold
higher than those obtained with native P-gp. The degree
of increase in the k
sub
⁄ K
m(D)
values with linker-cleaved
P-gp differed for each substrate. The k
sub
⁄ K
m(D)
value is
a measure of substrate specificity [32]. Thus, the
k
sub
⁄ K
m(D)
value for ATPase activity can be assumed to
represent the transport substrate specificity, and the
shifts in substrate specificity with the linker-cleaved
P-gp can be represented as the relative ratio of
the k
sub
⁄ K
m(D)
value between the native P-gp and the
protease-cleaved P-gp. The relative ratio of the
k

sub
⁄ K
m(D)
value between the native P-gp and the
protease-cleaved P-gp for each transport substrate is
shown in Fig. 6. The relative ratios of the k
sub
⁄ K
m(D)
values are < 100% because the k
sub
⁄ K
m(D)
values of
native P-gp are less than those of protease-cleaved P-gp.
With vinblastine, calcein-AM and valinomycin, the
relative ratios of the k
sub
⁄ K
m(D)
values between the
native P-gp and the protease-cleaved P-gp were
relatively high, with values ranging from 34% to 96%,
whereas with the other substrates, the relative ratios
were low and the values ranged from 6% to 15%. For
V8 protease-cleaved P-gp, the kinetic tendency described
above was similar to that of trypsin-cleaved P-gp and
chymotrypsin-cleaved P-gp (data not shown).
Discussion
We investigated the role of the linker region in human

P-gp that spans from approximately Glu633 to
Tyr709. As previously reported [21–24], the linker
region appears to be the most flexible part of the P-gp
structure (Fig. 1). We identified the cleavage sites of
trypsin, chymotrypsin and V8 protease as Arg680,
Leu662 and Glu652, respectively (Table 1). Nuti et al.
[22] identified the same trypsin cleavage site at Arg680,
but a different chymotrypsin cleavage site at Leu682.
This difference may be a result of different P-gp prepa-
rations; while our study used a purified preparation in
detergent micelles, Nuti et al. used crude membrane
fractions [22].
A comparison between native P-gp and linker-
cleaved P-gp indicated that the linker region of P-gp
seems to participate in neither the preservation of the
overall structure nor the ATPase reaction itself. This is
supported by the following findings. (i) Cleaving the
linker did not inactivate the ATPase activity of P-gp;
rather, linker-cleaved P-gp exhibited higher basal and
substrate-stimulated ATPase activity than native P-gp
(Fig. 2 and Table 2). These results indicate that, as for
the native P-gp, the N-half and the C-half fragments of
the linker-cleaved P-gp interact with each other during
ATP hydrolysis. In addition, when recombinant N-half
and C-half P-gp fragments were expressed alone, they
did not exhibit substrate-stimulated ATPase activity
[33]. (ii) Size-exclusion chromatography analysis indi-
cated that the N-half and C-half fragments of P-gp are
neither aggregated nor dissociated by cleavage of the
linker region (Fig. 3). This finding is further supported

by co-immunoprecipitation studies [21] and a pull-
down assay [34]. (iii) Cleavage of the linker did not
affect the thermostability of P-gp (Fig. 4).
Increases in the verapamil-stimulated ATPase activ-
ity correlated with a decrease in the residual amount
Rhodamine 123
Colchicine
Verapamil
Nicardipine
Rhodamine B
Digoxin
Vinblastine
Paclitaxel
Calcein-AM
Valinomycin
0
10
60
50
40
30
20
70
80
130
120
110
100
90
140

The relative ratios of k
sub
/K
m(D)
values
between native and protease cleaved P-gp
(% protease cleaved P-gp)
Fig. 6. Ratios of the k
sub
⁄ K
m(D)
values between native and prote-
ase-cleaved P-gp. The k
sub
⁄ K
m(D)
value of native P-gp was divided
by that of trypsin-cleaved (filled columns) or chymotrypsin-cleaved
(open columns) P-gp. The quotients are shown as a percentage.
Functional role of the linker region in P-gp T. Sato et al.
3510 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS
of native P-gp (Fig. 2), revealing that cleavage of the
P-gp linker region increased P-gp ATPase activity.
The k
sub
values for all of the tested substrates were
increased by linker cleavage (Table 2). Thus, the
increased ATPase activity with linker cleavage was
common for all the substrates tested. Moreover, the
substrate-stimulated ATPase activity was observed in

both protease-cleaved P-gp and native P-gp (Table 2),
indicating that linker-cleaved P-gp can recognize trans-
port substrates. Taken together, these data suggest
that the linker region regulates the ATP hydrolysis
rate. Thus, one possible role for the linker region is
that it serves as a cleavage activation site, as described
previously [21]. However, increased ATPase activity
also indicates that the linker region has another role
as a suppressor of ATPase activity in the native P-gp.
The k
sub
⁄ k
basal
values of native P-gp were higher and
ranged from 2.2 to 18, whereas those of linker-cleaved
P-gp were lower and ranged from 1.3 to 4.7 (Table 2).
Thus, the linker region appears to suppress the basal
ATPase activity rather than the substrate-stimulated
ATPase activity. The k
sub
⁄ k
basal
values of the half-size
ABC transporters, MsbA and Sav1866, ranged from 2
to 5, which are more similar to the linker-cleaved P-gp
values than to those of native P-gp [35–38]. Thus, the
linker region seems to be necessary to achieve high
k
sub
⁄ k

basal
values. The k
sub
⁄ k
basal
value is a ratio of
coupled to uncoupled ATPase activity with substrate
recognition, suggesting that the k
sub
⁄ k
basal
value repre-
sents the tightness of coupling between ATP hydro-
lysis and substrate recognition. Therefore, the linker
region of P-gp may increase the tightness of coupling
between ATP hydrolysis and substrate recognition and
contribute to efficient substrate recognition. The
involvement of the linker region in the coupling of
ATP hydrolysis with transport was suggested previ-
ously by Nuti et al. [22].
To investigate how cleavage of the P-gp linker
increased ATPase activity, we examined the crystal
structures of the inward-facing (closed apo) state and
outward-facing (nucleotide bound) state of MsbA [28],
a bacterial homolog of P-gp. The conformational
change between these two states is thought to regulate
the rate of ATP hydrolysis. This is because the forma-
tion of a canonical ATP dimer sandwich of the NBDs
and subsequent ATP hydrolysis occur in the outward-
facing state, and P

i
⁄ ADP release restores the inward-
facing state. The linker region of P-gp can be assumed
to connect the C-terminal helix of subunit A (shown in
red in Fig. 7) with the N-terminal elbow helix of sub-
unit B (shown in purple in Fig. 7) in the MsbA dimer.
In the inward-facing state, there appears to be less
interaction between the linker region and each subunit
because both ends of these two helices are exposed to
solvent. However, in the outward-facing state, the
N-terminal elbow helix is in closer proximity to the
plasma membrane, and the C-terminal a-helix moves
to the bottom center of the NBD dimer (Fig. 7B).
Thus, the linker region should pass around the NBD
surface, and there appears to be more interactions
between the linker region and subunit B because the
C-terminal a-helix comes into close proximity to the
NBD of subunit B. Therefore, during a conformational
change between the two states, the linker region might
interact with subunit B and cause steric hindrance.
Moreover, some interactions within the linker region
in the inward-facing state may need to be broken in
order to change the linker from a contracted to an
extended structure (Fig. 7A). Taken together, this
analysis indicates that because of interference of the
linker region, native P-gp cannot easily change its
conformation. However, in the absence of the linker
interference, the linker-cleaved P-gp can change
conformation more easily and exhibit increased
ATPase activity.

The analysis of the kinetic parameters for 10 trans-
port substrates indicated that linker cleavage modu-
lated the ATPase activity differently for each
substrate. With some transport substrates, several-fold
differences in the K
m(D)
values and a few dozen-fold
differences in the k
sub
⁄ K
m(D)
values were observed
between the native P-gp and the protease-cleaved P-gp
(Table 2). Thus, these data indicate that the linker
region affects P-gp substrate recognition, although
there seems to be no direct interaction between the
linker region and transport substrates. The k
sub
⁄ K
m(D)
of ATPase activity can be assumed to represent trans-
port substrate specificity. Thus, we evaluated the rela-
tive ratio of the k
sub
⁄ K
m(D)
values between the native
P-gp and the linker-cleaved P-gp to investigate the
effect of the linker region on P-gp substrate specificity.
With vinblastine, calcein-AM and valinomycin, the

relative ratios of the k
sub
⁄ K
m(D)
values between the
native P-gp and the linker-cleaved P-gp were higher
than those with the other substrates (Fig. 6). This
result indicates that native P-gp has relatively higher
substrate specificity for these three substrates than the
linker-cleaved P-gp. Therefore, the relative substrate
specificity is shifted by linker cleavage, suggesting
that the linker region enhances the inherent substrate
specificity of P-gp.
Transport measurement is needed to elucidate the
role of the linker in substrate export. A relatively
hydrophilic substrate, such as the peptide DAMGO
(Tyr-d-Ala-Gly-N-Methyl-Phe-Gly-ol) [39], would be
suitable for this measurement.
T. Sato et al. Functional role of the linker region in P-gp
FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3511
Materials and methods
Materials
L-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-
treated trypsin was purchased from Promega (Madison, WI,
USA). Chymotrypsin and V8 proteinase were purchased from
Roche Diagnostics (Mannheim, Germany). Subtilisin,
digoxin, nicardipine and calcein-AM were purchased from
Sigma (St Louis, MO, USA). Rhodamine 123, colchicine,
verapamil, rhodamine B, vinblastine and pacritaxel were
purchased from Wako (Osaka, Japan). Valinomycin was

purchased from Fluka (Buchs, Switzerland). n-Undecyl-b-d-
maltopyranoside (b-UDM) was purchased from Anatrace
(Maumee, OH, USA). Escherichia coli total lipid extract was
purchased from Avanti Polar Lipids (Alabaster, AL, USA).
Expression and purification of P-gp
Histidine-tagged wild-type human P-gp was expressed using
the baculovirus ⁄ expressSF+ insect cell system and purified
as described previously [27], with slight modifications.
Briefly, the expressSF+ membranes containing overexpres-
sed P-gp were solubilized with buffer containing 1.0%
b-UDM, and insoluble materials were removed by centrifu-
gation at 100 000 g for 1 h. The P-gp was purified by one-
step affinity chromatography using Talon Superflow Metal
Affinity Resin (Clontech, Mountain View, CA, USA) with
buffer containing 0.087% b-UDM. When necessary, the
purified P-gp was concentrated using an Amicon-Ultra
device with a molecular mass cut-off of 50 k (Millipore,
Bedford, MA, USA). The P-gp preparation had high pur-
ity, as shown in lane 1 of Fig. 1. All purification steps were
performed at 4 °C. The purified P-gp was stored at )80 °C
until further use.
Reconstitution into liposomes
To prepare liposomes, 50 mg of E. coli total lipid extract
dissolved in chloroform was dried and hydrated with
2.5 mL of ATPase reaction buffer (40 mm Tris–HCl, pH
7.4, 0.1 mm EGTA, 2 mm dithiothreitol). The hydrated
lipid suspension was subjected to five freeze–thaw cycles.
Frozen stocks of lipid were stored at )80 °C. After freeze–
*
*

C-terminal
α
-helix
Subunit A
Subunit B
*
*
NBD
TM
Inward facing state
A
B
(closed apo state)
Outward facing state
(nucleotide binding state)
90°
Subunit A
Subunit B
C-terminal
α
-helix
Elbow helix
domain
Fig. 7. Schematic diagrams of the P-gp
linker superimposed on the MsbA
structures. Two MsbA conformations in the
inward-facing (closed apo state, PDB ID;
3b5x) and outward-facing (nucleotide bound
state, PDB ID; 3b60) states are shown. One
monomer of the MsbA (subunit A) is shown

in light pink and the other (subunit B) is
shown in gray. (A) Side view of the
diagrams. The C-terminal a-helix of subunit
A is shown in red and the N-terminal elbow
helix of subunit B is shown in purple. The
putative linker region of P-gp is shown as a
dotted line and the start of the linker region
is denoted with an asterisk. The minimum
path of the linker region is shown as a blue
dotted line. The arrow represents the
movement of the N-terminal elbow helix of
subunit B during the conformational change
from the inward-facing state to the outward-
facing state. (B) Bottom-up view of the
NBDs: the diagrams shown in Fig. 7A were
rotated 90 ° around a horizontal axis. The
arrows represent the movement of each
NBD during the conformational change
from the inward-facing state to the outward-
facing state.
Functional role of the linker region in P-gp T. Sato et al.
3512 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS
thawing, the lipid suspension was sonicated in a bath soni-
cator until the suspension clarified. For reconstituting P-gp
into liposomes, purified P-gp (P-gp was purified using a
two-step procedure: TALON metal affinity and size-exclu-
sion chromatography) containing 0.06% b-UDM was
diluted 10-fold in the lipid-containing ATPase reaction
buffer at a protein : lipid ratio of 1 : 1 (w ⁄ w). Then, the
mixture was incubated at 23 °C for 20 min.

Protease treatment of purified P-gp in detergent
micelles
Purified P-gp (2 mgÆ mL
)1
) in detergent micelles [20 mm
piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES), pH 6.5;
300 mm NaCl, 300 mm imidazole, 20% glycerol, 0.087%
b-UDM, 0.1 mgÆmL
)1
of asolectin] was treated with trypsin,
chymotrypsin, V8 protease, or subtilisin at 37 °C for various
periods of time. The P-gp : trypsin, P-gp : chymotrypsin,
P-gp : V8 protease and P-gp : subtilisin ratios were 20 : 1,
20 : 1, 20 : 1 and 100 : 1 (w ⁄ w), respectively. The cleavage
was stopped by adding an excess of soybean trypsin inhibitor
(Roche) and 1 mm phenylmethanesulfonyl fluoride. The
cleaved P-gp was separated by SDS–PAGE and then visua-
lized with silver staining.
Measurement of trypsin cleavage kinetics of
reconstituted P-gp and its residual ATPase
activity
Purified and reconstituted P-gp (0.01 mgÆ mL
)1
) was incu-
bated with TPCK-treated trypsin at a P-gp : trypsin ratio
of 50 : 1 (w ⁄ w) at 20 °C for various periods of time. The
cleavage was stopped by addition of excess soybean trypsin
inhibitor (Roche) and 1 mm phenylmethanesulfonyl fluo-
ride. The cleaved P-gp was subjected to an ATPase assay in
the presence of 50 lm verapamil at 37 °C for 30 min. Then,

the samples were separated by SDS–PAGE and visualized
with silver staining.
N-terminal amino acid sequencing of
protease-cleaved P-gp
N-terminal amino acid sequence analysis of protease-
cleaved P-gp was performed under two conditions, as fol-
lows. Condition I (mild treatment with various proteases):
purified P-gp (2 mgÆmL
)1
) in buffer (20 mm PIPES, pH 6.5;
300 mm NaCl, 300 mm imidazole, 20% glycerol, 0.087%
b-UDM, 0.1 mgÆmL
)1
of asolectin) was incubated with
trypsin, chymotrypsin, or V8 protease at a P-gp : protease
ratio of 200 : 1 (w ⁄ w) at 20 °C for 30 min. Condition II
(extensive trypsin treatment): purified P-gp (3 mgÆmL
)1
)
in buffer (20 mm PIPES, pH 6.5; 300 mm NaCl, 300 mm
imidazole, 20% glycerol, 0.087% b-UDM, 0.1 mgÆmL
)1
of
asolectin, 5 mm MgATP) was incubated with trypsin at a
P-gp : trypsin ratio of 20 : 1 (w ⁄ w) at 37 °C for 30 min. In
both conditions, 15–30 lg of the digested fragments
were separated by SDS–PAGE and transferred to an
Immobilon-P transfer membrane (Millipore). The fragments
were stained with Coomassie Brilliant Blue R-350 (GE
Healthcare, UK Ltd), excised from the membranes and

analyzed using a Procise 492HT protein sequencer (Applied
Biosystems, Foster City, CA, USA). Although some sub
peaks were found, only the main peaks were unequivocally
interpretable and recorded as valid data.
Size-exclusion chromatography of native and
protease-cleaved P-gp
Purified P-gp (1 mgÆmL
)1
) in buffer (20 mm PIPES, pH
6.5; 300 mm NaCl, 300 mm imidazole, 20% glycerol,
0.087% b-UDM, 0.02% cholesteryl hemisuccinate) was
incubated with trypsin at a P-gp : trypsin ratio of 200 : 1
(w ⁄ w) at 20 °C for 30 min. Size-exclusion chromatography
was performed on a Superdex 200 10 ⁄ 300 GL column
(GE Healthcare) at 4 °C. The running buffer consisted of
20 mm PIPES (pH 6.5), 200 mm NaCl, 10% glycerol,
5mm dithiothreitol, 0.06% b-UDM and 0.02% cholesteryl
hemisuccinate, and the flow rate was 0.5 mL per min.
Each sample (100 lL) containing 100 lg of P-gp was
loaded, and the elution profiles were monitored by the
absorbance at 280 nm. This experiment was performed
twice.
ATPase measurements
The reconstituted protein (100–400 ng) was incubated in
20 lLof40mm Tris–HCl (pH 7.4) 0.1 mm EGTA, 2 mm
dithiothreitol, 5 mm MgATP and various concentrations of
transport substrates at 37 °C for 30 min. After the reaction,
the samples (16 lL) were mixed with 15 lL of 12% SDS to
stop the ATP hydrolysis reaction. The amount of released
inorganic phosphate was measured using a colorimetric

method [40]. All data points represent the means ± SD
from three independent assays. Error bars are shown unless
they are smaller than the symbol. The initial hydrolysis rate
was routinely calculated using a one-point assay at 30 min
because linearity in the time course was confirmed until
30 min within 37 lm per min of the initial rate (data not
shown). In the present study we performed the measure-
ments under conditions that restrict the initial rates below
this value.
SDS–PAGE analysis
Samples were prepared in 1 · buffer (10 mm Tris–HCl, pH
8.0; 10% sucrose, 40 mm dithiothreitol, 1 mm EDTA, 2%
SDS, 10 lgÆmL
)1
pyronin Y) and incubated at 50 °C for
15 min before electrophoresis. SDS–PAGE was performed
T. Sato et al. Functional role of the linker region in P-gp
FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3513
following the method of Laemmli [41] using PAGEL
(5–20%) (ATTO, Tokyo, Japan). Proteins separated in the
polyacrylamide gel were visualized using a Silver Stain II
Kit (Wako).
Measurement of protein concentrations
The protein concentration of purified P-gp was determined
by measuring the absorbance at 280 nm. The A
0.1%
value
at 280 nm was 0.76, which was calculated from the amino
acid composition [42]. Protein concentrations of cell lysates
and the crude membrane fractions were determined using

the bicinchoninic acid protein assay kit (Pierce, Rockford,
IL, USA) with BSA as the standard.
Kinetic parameters for the enzymatic reaction
of P-gp
For rhodamine 123, colchicine, verapamil, rhodamine B
and vinblastine, we used a modified Michaelis–Menten
equation (Eqn 1) proposed by Al-Shawi et al. [13]. The
rate of P-gp ATPase activity was expressed as follows:
v ¼ k
basal
e½þ
k
sub
À k
basal
ðÞe½s½
K
mðDÞ
þ s½

1 À
s½
K
i
þ s½

ð1Þ
where v is the ATPase activity, [s] is the substrate concen-
tration, [e] is the P-gp concentration, k
basal

is the basal
ATPase activity with no transport substrate, k
sub
is the
maximal ATPase activity associated with substrate activa-
tion, K
m(D)
is the apparent Michaelis constant for substrate
activation and K
i
is the inhibition constant for substrate
inhibition.
For nicardipine, digoxin, paclitaxel, calsein-AM and vali-
nomycin, we used a simple Michaelis–Menten equation
(Eqn 2). The rate of P-gp ATPase activity was expressed
as follows:
v ¼ k
basal
e½þ
k
sub
À k
basal
ðÞe½s½
K
mðDÞ
þ s½
ð2Þ
Fitting was carried out using grafit, version 5.04, soft-
ware (Erithacus Software, Staines, Middlesex, London, UK).

Illustration of the schematic diagrams of P-gp
linker superimposed on the MsbA structures
The schematic diagrams of two MsbA crystal structures in
the inward (closed apo state, PDB ID; 3b5x) and outward
(nucleotide bound state, PDB ID; 3b60) facing states [28]
were illustrated using PyMOL (). The
putative linker region of P-gp was superimposed on each
MsbA conformation by freehand drawing using Adobe
Illustrator CS.
Acknowledgements
This work was supported by Grants-in-aid for Scien-
tific Research from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan,
and by a Life Sciences Research Grant from the Take-
da Science Foundation. T.S. was supported by the 21st
Century COE Programs ‘Knowledge Information
Infrastructure for Genome Science’ and ‘Initiatives for
Attractive Education in Graduate Schools’ from
MEXT.
References
1 Ueda K, Cardarelli C, Gottesman MM & Pastan I
(1987) Expression of a full-length cDNA for the human
‘‘MDR1’’ gene confers resistance to colchicine, doxoru-
bicin, and vinblastine. Proc Natl Acad Sci USA 84,
3004–3008.
2 Gottesman MM & Pastan I (1993) Biochemistry of
multidrug resistance mediated by the multidrug trans-
porter. Annu Rev Biochem 62, 385–427.
3 Borst P, Schinkel AH, Smit JJ, Wagenaar E, Van
Deemter L, Smith AJ, Eijdems EW, Baas F & Zaman

GJ (1993) Classical and novel forms of multidrug resis-
tance and the physiological functions of P-glycoproteins
in mammals. Pharmacol Ther 60, 289–299.
4 Childs S & Ling V (1994) The MDR superfamily of
genes and its biological implications. In Important
Advances in Oncology (DeVita VT, Hellman S &
Rosenberg SA, eds), pp. 21–36. Lippincott Williams &
Wilkins, Philadelphia, PA.
5 Higgins CF (1992) ABC transporters: from microorgan-
isms to man. Annu Rev Cell Biol 8, 67–113.
6 Kimura Y, Morita SY, Matsuo M & Ueda K (2007)
Mechanism of multidrug recognition by
MDR1 ⁄ ABCB1. Cancer Sci 98 , 1303–1310.
7 Ambudkar SV, Cardarelli CO, Pashinsky I & Stein WD
(1997) Relation between the turnover number for vin-
blastine transport and for vinblastine-stimulated ATP
hydrolysis by human P-glycoprotein. J Biol Chem 272,
21160–21166.
8 Sarkadi B, Price EM, Boucher RC, Germann UA &
Scarborough GA (1992) Expression of the human mul-
tidrug resistance cDNA in insect cells generates a high
activity drug-stimulated membrane ATPase. J Biol
Chem 267, 4854–4858.
9 Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO,
Gottesman MM & Pastan I (1992) Partial purification
and reconstitution of the human multidrug-resistance
pump: characterization of the drug-stimulatable
ATP hydrolysis. Proc Natl Acad Sci USA 89, 8472–
8476.
10 Doige CA, Yu X & Sharom FJ (1992) ATPase activity

of partially purified P-glycoprotein from multidrug-
Functional role of the linker region in P-gp T. Sato et al.
3514 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS
resistant Chinese hamster ovary cells. Biochim Biophys
Acta 1109, 149–160.
11 Kimura Y, Kioka N, Kato H, Matsuo M & Ueda K
(2007) Modulation of drug-stimulated ATPase activity
of human MDR1 ⁄ P-glycoprotein by cholesterol.
Biochem J 401, 597–605.
12 Litman T, Zeuthen T, Skovsgaard T & Stein WD
(1997) Structure-activity relationships of P-glycoprotein
interacting drugs: kinetic characterization of their
effects on ATPase activity. Biochim Biophys Acta 1361,
159–168.
13 Al-Shawi MK, Polar MK, Omote H & Figler RA
(2003) Transition state analysis of the coupling of drug
transport to ATP hydrolysis by P-glycoprotein. J Biol
Chem 278, 52629–52640.
14 Aanismaa P & Seelig A (2007) P-Glycoprotein kinetics
measured in plasma membrane vesicles and living cells.
Biochemistry 46, 3394–3404.
15 Gatlik-Landwojtowicz E, Aanismaa P & Seelig A (2006)
Quantification and characterization of P-glycoprotein-
substrate interactions. Biochemistry 45, 3020–3032.
16 Chen CJ, Chin JE, Ueda K, Clark DP, Pastan I,
Gottesman MM & Roninson IB (1986) Internal
duplication and homology with bacterial
transport proteins in the mdr1 (P-glycoprotein)
gene from multidrug-resistant human cells. Cell 47,
381–389.

17 Germann UA, Chambers TC, Ambudkar SV, Licht T,
Cardarelli CO, Pastan I & Gottesman MM (1996)
Characterization of phosphorylation-defective mutants
of human P-glycoprotein expressed in mammalian cells.
J Biol Chem 271, 1708–1716.
18 Hrycyna CA, Ramachandra M, Germann UA, Cheng
PW, Pastan I & Gottesman MM (1999) Both ATP sites
of human P-glycoprotein are essential but not symmet-
ric. Biochemistry 38, 13887–13899.
19 Holland IB & Blight MA (1999) ABC-ATPases, adapt-
able energy generators fuelling transmembrane move-
ment of a variety of molecules in organisms from
bacteria to humans. J Mol Biol 293, 381–399.
20 Kusuhara H & Sugiyama Y (2007) ATP-binding cas-
sette, subfamily G (ABCG family). Pflugers Arch 453,
735–744.
21 Nuti SL, Mehdi A & Rao US (2000) Activation of the
human P-glycoprotein ATPase by trypsin. Biochemistry
39, 3424–3432.
22 Nuti SL & Rao US (2002) Proteolytic cleavage of the
linker region of the human P-glycoprotein modulates its
ATPase function. J Biol Chem 277, 29417–29423.
23 Wang G, Pincheira R, Zhang M & Zhang JT (1997)
Conformational changes of P-glycoprotein by nucleo-
tide binding. Biochem J 328 (Pt 3), 897–904.
24 Ghosh P, Moitra K, Maki N & Dey S (2006) Allo-
steric modulation of the human P-glycoprotein
involves conformational changes mimicking catalytic
transition intermediates. Arch Biochem Biophys 450,
100–112.

25 Rao PS, Mallya KB, Srivenugopal KS, Balaji KC &
Rao US (2006) RNF2 interacts with the linker region
of the human P-glycoprotein. Int J Oncol 29,
1413–1419.
26 Georges E (2007) The P-glycoprotein (ABCB1) linker
domain encodes high-affinity binding sequences to
alpha- and beta-tubulins. Biochemistry 46, 7337–7342.
27 Kodan A, Shibata H, Matsumoto T, Terakado K,
Sakiyama K, Matsuo M, Ueda K & Kato H (2009)
Improved expression and purification of human multi-
drug resistance protein MDR1 from baculovirus-
infected insect cells. Protein Expr Purif 66, 7–14.
28 Ward A, Reyes CL, Yu J, Roth CB & Chang G (2007)
Flexibility in the ABC transporter MsbA: Alternating
access with a twist. Proc Natl Acad Sci USA 104,
19005–19010.
29 Globisch C, Pajeva IK & Wiese M (2008) Identification
of putative binding sites of P-glycoprotein based on its
homology model. ChemMedChem 3, 280–295.
30 Muller M, Bakos E, Welker E, Varadi A, Germann
UA, Gottesman MM, Morse BS, Roninson IB & Sark-
adi B (1996) Altered drug-stimulated ATPase activity in
mutants of the human multidrug resistance protein.
J Biol Chem 271, 1877–1883.
31 Loo TW & Clarke DM (1999) Identification of residues
in the drug-binding domain of human P-glycoprotein.
Analysis of transmembrane segment 11 by cysteine-
scanning mutagenesis and inhibition by dibromobi-
mane. J Biol Chem 274, 35388–35392.
32 Copeland RA (2000) ENZYMES A Practical Introduc-

tion to Structure, Mechanism, and Data Analysis. 2nd
edn. Wiley-VCH, New York, NY.
33 Loo TW & Clarke DM (1994) Reconstitution of drug-
stimulated ATPase activity following co-expression of
each half of human P-glycoprotein as separate polypep-
tides. J Biol Chem 269, 7750–7755.
34 Loo TW & Clarke DM (1996) The minimum functional
unit of human P-glycoprotein appears to be a mono-
mer. J Biol Chem 271, 27488–27492.
35 Doerrler WT & Raetz CR (2002) ATPase activity of the
MsbA lipid flippase of Escherichia coli. J Biol Chem
277, 36697–36705.
36 Reuter G, Janvilisri T, Venter H, Shahi S, Balakrishnan
L & van Veen HW (2003) The ATP binding cassette
multidrug transporter LmrA and lipid transporter
MsbA have overlapping substrate specificities. J Biol
Chem 278, 35193–35198.
37 Eckford PD & Sharom FJ (2008) Functional charac-
terization of Escherichia coli MsbA: interaction with
nucleotides and substrates. J Biol Chem 283, 12840–
12850.
38 Velamakanni S, Yao Y, Gutmann DA & van Veen HW
(2008) Multidrug transport by the ABC transporter
T. Sato et al. Functional role of the linker region in P-gp
FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS 3515
Sav1866 from Staphylococcus aureus. Biochemistry 47,
9300–9308.
39 Oude Elferink RP & Zadina J (2001) MDR1 P-glyco-
protein transports endogenous opioid peptides. Peptides
22, 2015–2020.

40 Chifflet S, Torriglia A, Chiesa R & Tolosa S (1988) A
method for the determination of inorganic phosphate
in the presence of labile organic phosphate and high
concentrations of protein: application to lens ATPases.
Anal Biochem 168, 1–4.
41 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 277, 680–685.
42 Gill SC & von Hippel PH (1989) Calculation of protein
extinction coefficients from amino acid sequence data.
Anal Biochem 182, 319–326.
Supporting information
The following supplementary material is available:
Fig. S1. SDS–PAGE of P-gp after extensive trypsin
cleavage.
Fig. S2. Chemical structures of transport substrates
used in the present study.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Functional role of the linker region in P-gp T. Sato et al.
3516 FEBS Journal 276 (2009) 3504–3516 ª 2009 The Authors Journal compilation ª 2009 FEBS