Báo cáo khoa học: Dissection of LolB function – lipoprotein binding, membrane targeting and incorporation of lipoproteins into lipid bilayers potx

Dissection of LolB function – lipoprotein binding,
membrane targeting and incorporation of lipoproteins
into lipid bilayers
Jun Tsukahara, Keita Mukaiyama, Suguru Okuda, Shin-ichiro Narita and Hajime Tokuda
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan
Introduction
Lipoproteins represent a subset of proteins anchored
to membranes of both Gram-positive and Gram-nega-
tive bacteria. At least 90 species of lipoprotein are
found in Escherichia coli [1]. Lipoproteins are pro-
cessed to their mature forms on the outer leaflet of the
inner membrane [2], and then transported to the outer
membrane or retained in the inner membrane accord-
ing to the lipoprotein sorting signal located at position
2. Aspartic acid at this position functions as an inner
membrane retention signal, whereas other residues
function as outer membrane signals [3].
It has been found in E. coli that the sorting of
lipoproteins to the outer membrane is mediated by a
system composed of five Lol factors, LolA–LolE [3].
Keywords
lipoprotein; LolA; LolB; membrane targeting;
phospholipids
Correspondence
H. Tokuda, Institute of Molecular and
Cellular Biosciences, University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032,
Japan
Fax: +81 3 5841 8464
Tel: +81 3 5841 7830
E-mail:

(Received 1 May 2009, revised 1 June
2009, accepted 16 June 2009)
doi:10.1111/j.1742-4658.2009.07156.x
Escherichia coli cells express at least 90 species of lipoprotein. LolB is one
of the essential outer membrane lipoproteins, being involved in the last step
of lipoprotein sorting. It accepts lipoproteins from a periplasmic molecular
chaperone, LolA, and mediates the outer membrane anchoring of lipopro-
teins through a largely unknown mechanism. It has been shown previously
that a LolB derivative, mLolB, lacking an N-terminal acyl chain, can bind
lipoproteins. We examined how the lack of an N-terminal anchor affects
the outer membrane anchoring of lipoproteins. Surprisingly, mLolB com-
pensates for LolB function and supports E. coli growth, indicating that the
N-terminal anchor is not essential for its function. Indeed, mLolB correctly
localizes lipoproteins to either the inner or outer membrane depending on
the sorting signal at the steady state. Furthermore, periplasmic mLolB
enables the dissection of LolB function, namely lipoprotein binding, mem-
brane targeting and lipoprotein anchoring. It mediates the transfer of lipo-
proteins from LolA to the outer membrane, but also the inner membrane
and liposomes, indicating that mLolB exhibits no membrane preference
and targets to phospholipids. Consequently, an outer membrane-specific
lipoprotein is transiently mislocalized to the inner membrane when cells
express only mLolB. LolB anchored to the outer membrane does not cause
such mislocalization and is more active than mLolB. Phosphatidylethanol-
amine has been found to stimulate the mLolB-dependent membrane
anchoring of lipoproteins. Taken together, these results indicate that
lipoprotein binding, membrane targeting and membrane incorporation of
lipoproteins are intrinsic functions of LolB.
Abbreviations
CL, cardiolipin; IPTG, isopropyl thio-b-
D-galactoside; PE, phosphatidylethanolamine; PG, phosphatidylglycerol.

4496 FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS
The LolCDE complex in the inner membrane belongs
to the ATP-binding cassette transporter superfamily
and releases outer membrane-specific lipoproteins,
resulting in the formation of a soluble complex with a
periplasmic chaperone, LolA. Lipoproteins with aspar-
tic acid at position 2 are not recognized by LolCDE
and thus remain in the inner membrane. The LolA–
lipoprotein complex reaches the outer membrane via
the periplasm, and then interacts with a lipoprotein
receptor, LolB. LolB is itself a lipoprotein anchored to
the outer membrane, accepts a lipoprotein from LolA
and somehow incorporates it into the outer membrane.
The overall structures of LolA and LolB are very
similar [4]. They comprise 11 antiparallel b-strands,
which fold into an incomplete b-barrel, and two
loops covering the barrel. The barrel and the loops
containing three a-helices form a hydrophobic cavity,
which has recently been found to undergo opening
and closing on binding and release of lipoproteins,
respectively [5]. Lipoproteins are irreversibly trans-
ferred from LolA to LolB, because the hydrophobic
interaction with lipoproteins is stronger for LolB
than for LolA [6]. Moreover, the extra C-terminal
loop characteristic of LolA has been found to be
important for the prevention of the re-incorporation
of lipoproteins released from the inner membrane [7].
These observations reveal that LolA and LolB are
structurally similar, but play distinct roles in the
outer membrane sorting of lipoproteins. LolA func-

tion has been studied extensively because it is a solu-
ble protein. However, the function of LolB remains
largely unknown.
We found that mLolB lacking the N-terminal acyl
chain is functional. Taking advantage of this soluble
version of LolB, LolB function was dissected. It was
found that mLolB catalyses the membrane incorpora-
tion of lipoproteins.
Results
Membrane anchor of LolB is dispensable
It has been found previously that a LolB derivative,
mLolB, lacking an N-terminal lipid anchor, can accept
lipoproteins from LolA [8], but it is not known
whether mLolB can compensate completely for the
essential LolB function. To address this issue, E. coli
KT6 (DlolB::kan pKT021) [9] cells were further trans-
formed with either pYKT122 encoding LolB or
pYKT123 encoding mLolB under the control of the
arabinose promoter, and grown at 42 °C. The KT6
strain lacks the chromosomal lolB gene and harbours
pKT021 carrying a temperature-sensitive replicon and
lolB. This strain cannot grow at 42 °C because of the
deletion of pKT021 [9]. If LolB function is expressed
from the transformed plasmid, the strain will grow at
42 °C even after curing of the temperature-sensitive
plasmid pKT021. The strains thus obtained were
named KT60(DlolB::kan) ⁄ pYKT122 and KT60 ⁄ pYKT
123. Surprisingly, both KT60 ⁄ pYKT122 (LolB) and
KT60 ⁄ pYKT123 (mLolB) grew at 42 °C when arabi-
nose was added to the culture (Fig. 1), indicating

that the acyl chain anchor is dispensable for LolB
function.
To determine the minimum amounts of LolB and
mLolB required for growth, KT60 cells harbouring
pYKT122 or pYKT123 were grown in the presence of
various concentrations of arabinose (Fig. 2A). The
concentration of arabinose required to support normal
growth was slightly lower with LolB (0.002%) than
with mLolB (0.005%). The amounts of LolB and
mLolB expressed under the respective conditions were
determined by immunoblotting with anti-LolB serum,
which had been raised against purified LolB. For the
detection of LolB and mLolB, blotted membranes were
treated with an enhanced chemiluminescence substrate,
followed by detection with a lumino-image analyser as
described in Experimental procedures. The density of
mLolB expressed in the presence of 0.005% arabinose
was significantly lower than that of LolB expressed in
Time (h)
10
–2
10
–1
10
0
10
1
10
2
10

3
10
4
A
660
2468100
Fig. 1. mLolB can support E. coli growth. E. coli KT6 (DlolB:kan
pKT021) cells were transformed with pYKT122 carrying lolB
(squares) or pYKT123 carrying mlolB (triangles) under the control of
P
BAD
, or an empty vector pMAN885EH (circles), and grown at
42 °C in the presence (filled symbols) and absence (open symbols)
of 0.2% arabinose for the indicated times by the inoculation of
portions of cultures into fresh medium.
J. Tsukahara et al. Dissection of LolB function
FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS 4497
the presence of 0.002% arabinose (Fig. 2B). If the
immunodetection system accurately indicates the
amounts of LolB and mLolB, mLolB should be func-
tionally more active than LolB. However, this was not
the case, as the same amounts of purified LolB and
mLolB exhibited significantly different densities with
this detection system (Fig. 2B, right two lanes), indicat-
ing that acyl chains affect the immunodetection system.
Various amounts of purified LolB and mLolB
were analysed by SDS–PAGE and visualized with the
immunodetection system (Fig. 2C). Quantitative deter-
mination of the band densities revealed that the
amount of mLolB was underestimated by a factor of

about eight. It was then found that Tween 20 used to
decrease nonspecific bands caused the release of
mLolB from blotted membranes. We therefore re-esti-
mated the minimum amounts of LolB and mLolB
required for growth with this immunodetection system
using purified LolB and mLolB as standards. The min-
imum amount of mLolB was found to be more than
two-fold higher than that of LolB, indicating that the
lack of acyl chains decreases LolB activity.
Membrane localization of lipoproteins in cells
grown with mLolB
In order to confirm that mLolB is localized only in the
periplasm, KT60 cells harbouring pYKT122 or
pYKT123 were induced with 0.2% arabinose, fraction-
ated and then subjected to SDS–PAGE and immuno-
blotting (Fig. 3). Although LolB was localized only in
LolB
mLolB
10
–1
10
0
10
1
10
2
10
3
10
–2

0246810 0246810
Time (h)
A
660
mLolB
LolB
0
0.0002
0.0005
0.001
0.002
0.005
0.01
0.02
Ara (%)
LolB
mL
olB
LolB
0
0.0002
0.0005
0.001
0.002
0.005
0.01
0.02
0
1
2

3
4
5
0510
Density (arbitrary units)
LolB
mLolB
Amount (n
g
)
21.5
36.5
LolB
mLolB
(kDa)
0
0.0002
0.0005
0.001
0.002
0.005
0.01
0.02
Arabinose (%)
A
B
C
Fig. 2. Determination of the minimum amounts of LolB and mLolB
required for growth. (A) KT60 cells harbouring pYKT122 (left) or
pYKT123 (right) were grown overnight at 37 °C in the presence of

0.2% arabinose. The cells were harvested, washed with fresh med-
ium and then grown at 37 °C for the indicated times in the pres-
ence of the indicated concentrations of arabinose by inoculation of
portions of the cultures into fresh medium. (B) The same amounts
of cells grown in the presence of various concentrations of arabi-
nose for 11 h were analysed by SDS–PAGE, followed by visualiza-
tion with an immunodetection system, as described in
Experimental procedures. As controls, purified LolB and mLolB
(each 5 ng) were also analysed (right two lanes). (C) The indicated
amounts of purified LolB and mLolB were analysed by SDS–PAGE
with an immunodetection system, as described in (B). The densi-
ties of the bands were determined and plotted as a function of the
amounts of LolB and mLolB. LolB and mLolB (each 1 lg) were
analysed by SDS–PAGE, followed by staining with Coomassie bril-
liant blue (inset). The two proteins migrated to almost the same
position in SDS–PAGE.
LolB
SecB
MBP
SecG
WC
M
C
P
WC
M
C
P
LolB
mLolB

p
m
Fig. 3. mLolB is exclusively localized to the periplasm.
KT60 ⁄ pYKT122 (LolB) and KT60 ⁄ pYKT123 (mLolB) cells were
grown on LB medium supplemented with 0.2% arabinose at 37 °C.
Cells were harvested at a culture absorbance of 0.8 and fraction-
ated as described in Experimental procedures. Equivalent amounts
of the respective fractions were analysed by SDS–PAGE and visual-
ized with an immunodetection system, as described in Fig. 2. C,
cytoplasm; M, total membranes; P, periplasm; WC, whole cells.
The precursor (p) and mature (m) forms of mLolB are indicated at
the right of the gel. MBP, maltose-binding protein.
Dissection of LolB function J. Tsukahara et al.
4498 FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS
the total membrane fraction, the mature form of
mLolB was exclusively detected in the periplasm,
together with maltose-binding protein. The precursor
form of mLolB was found in the cytoplasmic fraction
in which SecB was present. An inner membrane
protein, SecG, was detected in the total membrane
fraction. These results indicate that mLolB lacking
the N-terminal acyl chain anchor is exclusively
localized in the periplasm and is able to support the
growth of cells.
Total membranes prepared from KT60 ⁄ pYKT123
cells grown with mLolB were further fractionated into
inner and outer membranes by sucrose density gradient
centrifugation, and analysed by SDS–PAGE, followed
by immunoblotting (Fig. 4A). OmpA and SecG were
examined as markers of the outer and inner mem-

branes, respectively. Four outer membrane-specific
lipoproteins, Lpp, Pal, BamD (formerly YfiO) and
LptE (formerly RlpB), were correctly localized in the
outer membrane, whereas the inner membrane-specific
AcrA remained in the inner membrane. Therefore,
lipoproteins are localized in the correct membranes in
the steady state, even when mLolB functions in the
periplasm.
To examine the sorting of lipoproteins in more
detail, KT60 cells growing with LolB or mLolB were
pulse labelled, and the membranes were fractionated
into inner and outer forms (Fig. 4B).
35
S-Labelled Lpp
was detected only in the outer membrane of cells
grown with LolB (left panel). In marked contrast, an
appreciable portion of
35
S-labelled Lpp was mislocal-
ized to the inner membrane of cells grown with mLolB
(middle panel). This mislocalized Lpp was quickly
chased to the outer membrane on incubation with non-
radioactive amino acids (right panel), indicating that
mLolB delivers lipoproteins to both the outer and
inner membranes.
In vitro membrane targeting activity of mLolB
To examine whether mLolB distinguishes between the
inner and outer membranes,
35
S-labelled Lpp released

with LolA from spheroplasts was isolated and
incubated at 30 °C for 30 min with outer membranes
prepared from LolB-depleted cells or inner mem-
branes in the presence of the specified concentrations of
mLolB (Fig. 5A). The reaction mixtures were
fractionated into pellets and supernatants to examine
35
S-labelled Lpp. Essentially all
35
S-labelled Lpp
remained in the supernatants when mLolB was not
added. The amount of
35
S-labelled Lpp in the pellet
fraction increased with an increase in the amount of
added mLolB. Moreover, mLolB exhibited no mem-
brane preference and caused incorporation of Lpp into
both the outer and inner membranes.
We then examined the mLolB-dependent localization
of lipoproteins to liposomes prepared from E. coli phos-
pholipids (Fig. 5B). Because of the technical difficulty in
preparing a large amount of LolA–[
35
S]Lpp complex,
the nonlabelled LolA–Pal complex was obtained as a
spheroplast supernatant after the LolA-dependent
release assay, and incubated with liposomes in the pres-
ence and absence of mLolB. Almost all Pal molecules
were recovered in the liposome fraction after incubation
with liposomes and mLolB. In contrast, Pal remained

soluble when either mLolB or liposomes were omitted.
The amount of Pal incorporated into liposomes was
determined and plotted as a function of time (Fig. 5C).
Taken together, these results indicate that mLolB
targets and transfers lipoproteins to the lipid bilayer.
OmpA
SecG
Lpp
Pal
BamD
LptE
AcrA
OM IM
IMOM
IMOM
OmpA
SecG
Lpp
IMOM
LoIB
mLoIB
–
+
chase
–
A
B
Fig. 4. Sorting signal-specific membrane localization of lipoproteins
by mLolB. (A) KT60 ⁄ pYKT123 cells were grown on LB medium
supplemented with 0.2% arabinose at 37 °C. The cells were con-

verted into spheroplasts and disrupted as described in Experimental
procedures. The total membrane fractions were separated into
inner and outer membranes by sucrose density gradient centrifuga-
tion, followed by fractionation. Each fraction was analysed by SDS–
PAGE and immunoblotting with the indicated antibodies. (B)
KT60 ⁄ pYKT122 (LolB) and KT60 ⁄ pYKT123 (mLolB) cells were
grown on M63 (NaCl)-minimal medium and labelled with Tran[
35
S]-
label for 30 s. Where specified, labelling was chased by the addi-
tion of nonradioactive methionine and cysteine, as described in
Experimental procedures. The labelled cells were converted into
spheroplasts, and the total membrane fractions obtained on cell
disruption were fractionated into inner and outer membranes by
sucrose density gradient centrifugation, as described in (A).
J. Tsukahara et al. Dissection of LolB function
FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS 4499
Phosphatidylethanolamine (PE) is important for
LolB function
Escherichia coli membranes contain PE, phosphatidyl-
glycerol (PG) and cardiolipin (CL) as major phospho-
lipids. It has been found previously that the correct
sorting of lipoproteins at the release step involving
LolCDE is affected significantly by the phospholipid
composition, and that a nonbilayer phospholipid, PE,
is especially important [10,11]. The effect of phospho-
lipid composition on the mLolB-dependent localization
of Pal was examined with liposomes prepared from CL
or PG, with or without PE (Fig. 6A). Both the rate
and extent of mLolB-dependent incorporation of Pal

Input
0 0.5 1.0 2.0
mLolB (ng·mL
–1
)
2.00 0.5 1.0 2.0
OM (
Δ
LolB)
IM
ppt
sup
Membrane
None
0
20
40
60
80
100
0102030
Pal incorporated (%)
Time (min)
Pal
pspspspspspspspspsInput
0 30 0 30 0 3051020
+– +
+–+
mLolB
Liposome

Time (min)
A
B
C
Fig. 5. mLolB targets and transfers lipoproteins to the lipid bilayer.
(A) Spheroplasts prepared from MC4100 cells were labelled with
Tran[
35
S]label in the presence of hexahistidine-tagged LolA, followed
by isolation of the LolA–[
35
S]Lpp complex, as described in Experi-
mental procedures. The LolA–[
35
S]Lpp complex was then incubated
at 30 °C for 30 min with outer membranes (OM) prepared from
LolB-depleted cells, inner membranes (IM) or no membranes in the
presence of the indicated concentrations of mLolB. The reaction mix-
tures were fractionated into pellets and supernatants, which were
subjected to SDS–PAGE, followed by fluorography. (B) A spheroplast
supernatant was prepared as described in (A), except for the
Tran[
35
S]label. The spheroplast supernatants containing LolA–lipo-
protein complexes were then incubated with or without 100 lgÆmL
)1
liposomes prepared from E. coli phospholipids and 0.26 lgÆmL
)1
mLolB for the indicated times. The reaction mixtures were fraction-
ated into pellet (p) and supernatant (s) fractions and analysed by

SDS–PAGE and immunoblotting with anti-Pal serum. (C) The results
shown in (B) were quantified and plotted as a function of the
reaction time, taking the total amount of Pal as 100%.
pspspspspspsps
–
+
PG
PG/PE
pspspspspspsps
0300 305 10 20 0 30 0 3051020
–+
mLolB
Time
(min)
CL
CL/PE
A
B
mLolB
ps
psps ps
Pal incorporated (%)
0
20
40
60
80
pspsps psps
100
0

75
25
50
50
25
75
0
100
PG
CL
100
0
75
25
50
50
25
75
PG
PE
psps ps ps
100
0
75
25
50
50
25
75
CL

PE
–
+
100
0
75
25
50
50
25
75
0
100
PG
CL
100
0
75
25
50
50
25
75
PG
PE
100
0
75
25
50

50
25
75
CL
PE
Time (min)
Incorporation of pal (%)
0
20
40
60
80
100
0 102030
PG/PE
CL/PE
CL
PG
Fig. 6. PE stimulates the mLolB-dependent membrane incorpora-
tion of lipoproteins. (A) The incorporation of Pal into liposomes was
examined at 30 °C for the specified times, as in Fig. 5B, with sphe-
roplast supernatants containing the LolA–Pal complex. Liposomes
were prepared from CL or PG alone, or their mixture with PE added
to 50%, as indicated. Where specified, mLolB was not added. The
amounts of Pal incorporated into liposomes were determined and
calculated as described in Fig. 5B, C, and plotted as a function of
time. (B) Liposomes were prepared from the indicated combinations
of phospholipids mixed in various proportions (%). The incorporation
of Pal into these liposomes was examined at 30 °C for 10 min in
the presence and absence of mLolB. The amounts of Pal incorpo-

rated into liposomes were calculated as described in Fig. 5C.
Dissection of LolB function J. Tsukahara et al.
4500 FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS
into CL or PG liposomes were increased when PE
comprised 50% of the phospholipid.
The mLolB-dependent incorporation of Pal into lipo-
somes containing various combinations of phospholipid
was examined at 30 °C for 10 min (Fig. 6B). The incor-
poration of Pal into liposomes prepared from CL and
PG remained low irrespective of their proportions.
However, the incorporation of Pal into both CL and
PG liposomes increased with an increase in the propor-
tion of PE. PE possesses a small headgroup relative to
acyl chains and is known to be a nonbilayer phospho-
lipid. It causes curvature stress in the membrane and
affects the lateral pressure in the lipid bilayer. Such a
property of PE appears to be important for the mLolB-
dependent incorporation of lipoproteins.
LolA is essential even in the presence of mLolB
As the overall structures of LolA and LolB are very
similar [4], it was possible that periplasmic mLolB
might compensate for LolA function. To address this
issue, TT016 (lacPO-lolA) cells were transformed with
pMAN885EH (vector), pMAN995 (LolA), pYKT122
(LolB) or pYKT123 (mLolB). Because LolA is essen-
tial, TT016 cells did not grow in the absence of isopro-
pyl thio-b-d-galactoside (IPTG) unless LolA was
expressed from the plasmid (Fig. 7A). However,
expression of LolB or mLolB did not support the
growth of TT016 in the absence of IPTG.

We next examined whether mLolB can release Lpp
from spheroplasts (Fig. 7B).
35
S-Labelled Lpp
expressed in spheroplasts was almost completely
released into the spheroplast supernatant by the addi-
tion of LolA, whereas essentially all Lpp molecules
remained in the spheroplasts in the absence of LolA.
The addition of mLolB did not cause the release of
Lpp.
Taken together, these results indicate that mLolB
cannot compensate for LolA function.
Discussion
LolB is a lipoprotein anchored to the outer membrane
and catalyses the last step of lipoprotein sorting to the
outer membrane. We therefore expected that its acyl
chain anchor would significantly contribute to its func-
tion. However, the acyl chain anchor was found not to
be essential when mLolB was expressed in the peri-
plasm (Fig. 1). Newly synthesized Lpp in cells growing
in the presence of mLolB was transiently mislocalized
to the inner membrane (Fig. 4). This mislocalization
did not cause appreciable inhibition of growth,
although the concentration of mLolB required was
higher than that of LolB (Fig. 2). As the mislocaliza-
tion of Lpp to the inner membrane is highly toxic to
cells [12], mislocalized Lpp should be immediately
released from the inner membrane by LolCDE and
eventually localized to the outer membrane, from
which lipoproteins are not released. The N-terminal

anchor of LolB is therefore important for the outer
membrane-specific incorporation of lipoproteins. Fur-
thermore, as LolB is located more closely to mem-
branes than is periplasmic mLolB, membrane targeting
of LolB should occur more efficiently than that of
mLolB. These differences appear to cause the higher
activity of LolB than mLolB.
Because of the extra C-terminal loop, LolA cannot be
targeted to membranes [7], whereas membrane targeting
and subsequent lipoprotein incorporation were found to
be intrinsic functions of LolB. It is now clearly estab-
B
Lpp
ps
–
LolA-His mLolB
psps
0246
Vector
LolA
mLolB
LolB
10
–1
10
0
10
1
10
2

10
3
A
660
A
Time (h)
Fig. 7. mLolB does not compensate for LolA function. (A) E. coli
TT016 (lacPO-lolA) cells were transformed with pMAN885EH
(empty vector), pMAN995 (LolA), pYKT122 (LolB) or pYKT123
(mLolB), and then grown overnight at 37 °C on LB medium supple-
mented with 1 m
M IPTG. The cells were harvested, washed three
times with fresh LB medium and then grown at 37 °C on LB med-
ium supplemented with 0.02% arabinose for the specified times.
(B) The release of
35
S-labelled Lpp from spheroplasts was exam-
ined in the presence of LolA or mLolB, as described in Experimen-
tal procedures.
35
S-Labelled Lpp in pellet (p) and supernatant (s)
fractions was examined by SDS–PAGE and fluorography.
J. Tsukahara et al. Dissection of LolB function
FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS 4501
lished that LolA and LolB play distinct roles in the
sorting of lipoproteins, although the two proteins are
structurally very similar. Indeed, LolA is still essential
when mLolB is expressed in the periplasm (Fig. 7). The
function of LolA has been extensively studied in vitro
because it can be purified as a soluble protein. However,

LolB is anchored to the outer membrane, and its solubi-
lization requires a detergent. Because of this, it was not
feasible to examine whether the membrane incorpora-
tion of lipoproteins is an intrinsic function of LolB. It is
now clear that LolB function can be examined with a
soluble derivative, mLolB.
Our previous studies did not completely exclude the
possibility that an unknown factor might be present in
the outer membrane and be involved in the membrane
incorporation of lipoproteins. However, as mLolB was
found to be able to catalyse the incorporation of lipo-
proteins, even into liposomes, no extra factor is required
for the final step of lipoprotein sorting. The next ques-
tion is how mLolB is targeted to the membrane. We
have speculated previously that a loop protruding from
the LolB molecule might be important for membrane
targeting, because a hydrophobic residue located in the
loop appears to be adequate for this [4]. Derivatives of
mLolB defective in membrane targeting function are
currently under examination. The molecular mecha-
nisms underlying the LolB-dependent membrane incor-
poration of lipoproteins will be examined in detail
based on the crystal structure of mLolB derivatives.
The phospholipid composition significantly affects
the release of lipoproteins from the inner membrane
[11]. PE is critically important for the correct sorting of
lipoproteins, and PG suppresses the nonspecific release
of lipoproteins. However, the release of lipoproteins by
LolCDE from proteoliposomes reconstituted with CL
alone is completely independent of the sorting signal

[10,11]. PE enhances both the rate and extent of lipopro-
tein incorporation by mLolB (Fig. 6). PE has a small
headgroup relative to acyl chains and is known to affect
the lateral pressure in the lipid bilayer. As lipoproteins
contain three acyl chains derived from phospholipids
[13], their incorporation into the lipid phase is likely to
be affected by the nonbilayer property of PE.
Experimental procedures
Materials
Escherichia coli phospholipids were obtained from Avanti
Polar Lipids (Alabaster, AL, USA) and were washed with
acetone as reported previously [14]. Synthetic phospholip-
ids, CL, PG and PE, containing dioleoyl acyl chains (18:1,
9cis), were also obtained from Avanti Polar Lipids.
TALON Co
2+
affinity resin (Clontech, Mountain View,
CA, USA) was used to purify hexahistidine-tagged proteins.
Antibodies against LolA and Lpp were raised in rabbits as
described previously [15]. Tran[
35
S]label (mixture of 70%
[
35
S]Met and 20% [
35
S]Cys, 37 TBqÆmmol
)1
) was obtained
from MP Biochemicals. IgG sorb was purchased from

Enzyme Center Inc. (Boston, MA, USA) Sucrose monocap-
rate and n-dodecyl-b-d-maltopyranoside were purchased
from Dojindo Laboratories (Kumamoto, Japan).
Bacteria and media
KT60 (DlolB::kan) is a strain derived from KT6 (DlolB::kan
pKT021) [9] by curing pKT021, which carries bla, lolB and
a temperature-sensitive replicon, and always harbours a
specified plasmid carrying a functional LolB derivative. To
construct KT60, KT6 cells were transformed with the speci-
fied plasmid, and grown on Luria–Bertani (LB) medium
containing 25 lgÆmL
)1
chloramphenicol and 0.2% arabi-
nose at 42 °C for 9.5 h, followed by cultivation on LB
plates containing 0.2% arabinose. Ampicillin-sensitive cells
were isolated, and curing of pKT021 was confirmed. KT50
cells lack the major outer membrane lipoprotein Lpp and
were constructed from KT5 (DlolB::kan lpp pKT021) [9] by
substitution of pKT021 with pYKT123, as in the case of
the KT60 strain. TT016 (lacPO-lolA) [16] was used to
examine whether mLolB compensates for LolA function.
This strain carries the chromosomal lolA gene under the
control of the lactose promoter-operator and requires IPTG
for growth. MC4100 [17] was used to prepare spheroplasts
to examine lipoprotein release. Cells were grown on LB
broth (Difco, Sparks, MD, USA) or M63 (NaCl)-maltose
minimal medium [12]. When required, chloramphenicol was
added at 25 lgÆmL
)1
. The growth of E. coli cells was

followed by monitoring the absorbance at 660 nm.
Plasmids
To construct pYKT122 carrying lolB under the control of
P
BAD
,anEcoRI-HindIII fragment of pYKT100 [18] was
inserted into the same sites of pMAN885EH [12]. To con-
struct pYKT123 carrying mlolB under the control of P
BAD
,
a KpnI-HindIII fragment of pYKT102 [8] carrying the gene
for mlolB fused to the OmpF signal peptide was inserted
into the same sites of pMAN885EH.
Subcellular fractionation
KT60 cells harbouring pYKT122 or pYKT123 were grown
on M63 (NaCl)-maltose minimal medium supplemented
with 0.2% arabinose. Cells were harvested at a culture
absorbance of 0.8 and then converted into spheroplasts
according to the reported method [15]. The spheroplast
supernatant obtained on centrifugation at 10 000 g for
Dissection of LolB function J. Tsukahara et al.
4502 FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS
2 min was further centrifuged at 100 000 g for 1 h to
obtain a periplasmic fraction as a supernatant. Spheroplasts
were disrupted by sonication and centrifuged at 10 000 g
for 5 min to remove unbroken cells. The supernatant was
further centrifuged at 100 000 g for 1 h to obtain cyto-
plasmic and membrane fractions.
Separation of the inner and outer membranes
KT60 cells harbouring pYKT123 were converted into sphe-

roplasts as described above, and then disrupted by passage
through French pressure cells. After the removal of unbro-
ken cells by centrifugation at 10 000 g for 10 min, the total
membrane fraction obtained on centrifugation at 100 000 g
for 1 h was further separated by 30–55% (w ⁄ w) sucrose
density gradient centrifugation at 45 000 g for 14 h.
Pulse-chase experiment
KT60 cells harbouring pYKT122 or pYKT123 were grown
on M63 (NaCl)-maltose minimal medium supplemented
with 0.2% arabinose, 20 lgÆmL
)1
thiamine, 40 lgÆmL
)1
thymine, 40 lgÆmL
)1
uracil and 40 lg Æ mL
)1
each of all
amino acids except methionine and cysteine at 37 °C. When
the culture absorbance reached 0.8, the cells were labelled
with 1.85 MBq of Tran[
35
S]label for 30 s. Where indicated,
labelling was followed by a chase with nonradioactive
methionine and cysteine, each at 12 mm. The labelled cells
were immediately chilled by the addition of crushed ice,
converted into spheroplasts and then disrupted by sonica-
tion. After removal of unbroken cells by centrifugation at
10 000 g for 5 min, membrane fractions were obtained by
centrifugation at 100 000 g for 30 min, and then fraction-

ated by 30–55% (w ⁄ w) sucrose density gradient centrifuga-
tion as described above.
Preparation of LolB-depleted outer membranes
Outer membranes were prepared from KT50 cells harbour-
ing pYKT123 as described previously [15].
Purification of mLolB
MC4100 cells harbouring pYKT102 were grown at 37 °C
on LB medium containing 50 lgÆmL
)1
ampicillin. When the
culture absorbance reached 0.6, the expression of mLolB
was induced by the addition of 1 mm IPTG for 2 h. Peri-
plasmic fractions were prepared as described above and
then applied to a cation-exchange MonoS column (GE
Healthcare, Uppsala, Sweden), which had been equilibrated
with 25 mm sodium acetate, pH 5.0. The column was devel-
oped with a linear gradient of NaCl (0–1 m). The fractions
containing mLolB were collected and dialysed against
25 mm Tris ⁄ HCl, pH 8.2, followed by purification on an
anion-exchange MonoQ column (GE Healthcare) equili-
brated with 25 mm Tris ⁄ HCl, pH 8.2. The column was
developed with a linear gradient of NaCl (0–1 m).
Release of lipoproteins from spheroplasts
MC4100 cells were grown on M63 (NaCl)-maltose mini-
mal medium supplemented with 20 lgÆmL
)1
thiamine, 40
lgÆmL
)1
thymine, 40 lgÆmL

)1
uracil and 40 lgÆmL
)1
each of
all amino acids except methionine and cysteine. The cells
were converted into spheroplasts and then labelled with
0.37 MBq Tran[
35
S]label for 2 min at 30 °C in the presence
of 9.5 l gÆ mL
)1
hexahistidine-tagged LolA or mLolB, as
described previously [15]. After a 2 min chase with a 12 mm
nonradioactive methionine and cysteine mixture, the sphero-
plast suspension was chilled in ice–water, followed by centri-
fugation at 16 000 g for 2 min. The spheroplasts and
supernatant thus obtained were subjected to trichloroacetic
acid precipitation and then immunoprecipitation with anti-
Lpp serum, as reported previously [15]. [
35
S]-Labelled Lpp
was analysed by SDS–PAGE and fluorography.
mLolB-dependent membrane incorporation of
lipoproteins
The LolA–[
35
S]Lpp complex obtained in the spheroplast
supernatant after the lipoprotein release assay, as described
above, was adsorbed to TALON affinity resin and eluted
with 20 mm Tris ⁄ HCl, pH 7.5, containing 0.3 m NaCl and

0.25 m imidazole. After dilution with 20 m m Tris ⁄ HCl,
pH 7.5, the LolA–[
35
S]Lpp complex (100 lL) was incubated
with either outer or inner membranes (0.2 mgÆmL
)1
)at
30 °C for 30 min in the presence of various concentrations
of mLolB. The reaction mixture was transferred to ice and
centrifuged at 160 000 g for 1 h. [
35
S]Lpp in the pellet and
supernatant was analysed by SDS–PAGE and fluorogra-
phy, as reported previously [18]. Where specified, a nonla-
belled spheroplast supernatant containing the LolA–Pal
complex was also used to examine the mLolB-dependent
incorporation of Pal into liposomes.
SDS–PAGE and immunoblotting
SDS–PAGE was carried out according to Laemmli [19] or,
in the case of Lpp, Hussain et al. [20]. Immunoprecipitation
was carried out as described previously [15]. Proteins
labelled with Tran [
35
S] label were analysed by SDS–PAGE,
followed by fluorography with Enlightning (NEN Life Sci-
ence Products, Inc., Boston, MA, USA). To determine the
in vivo levels of LolB and mLolB, blotted poly(vinylidene
difluoride) membranes were treated with an enhanced
chemiluminescence substrate (ECL-Plus; GE Healthcare),
followed by detection with a lumino-image analyser (LAS-

1000plus; Fujifilm, Tokyo, Japan).
J. Tsukahara et al. Dissection of LolB function
FEBS Journal 276 (2009) 4496–4504 ª 2009 The Authors Journal compilation ª 2009 FEBS 4503
Other methods
Hexahistidine-tagged LolA was purified from TT015 [21]
cells harbouring pMAN995, as described previously [16].
Liposomes were prepared with a Mini-extruder (Avanti
Polar Lipids). Protein was determined by the method of
Lowry et al. [22] using bovine serum albumin as a standard.
Acknowledgements
We wish to thank Ms Naoko Yokota, University of
Tokyo, for the construction of pYKT122 and
pYKT123, and Dr Akihito Yamaguchi, Osaka Univer-
sity, for the anti-AcrA serum. This work was
supported by grants to H.T. from the Ministry of
Education, Science, Sports and Culture of Japan.
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