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

LolB is one of the essential outer membrane lipoproteins, being involved in the last step of lipoprotein sorting.. Results Membrane anchor of LolB is dispensable It has been found previo

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 sortaccord-ing 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: htokuda@iam.u-tokyo.ac.jp

(Received 1 May 2009, revised 1 June

2009, accepted 16 June 2009)

doi:10.1111/j.1742-4658.2009.07156.x

Escherichia colicells 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.

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 42C The KT6

strain lacks the chromosomal lolB gene and harbours

pKT021 carrying a temperature-sensitive replicon and

lolB This strain cannot grow at 42C because of the deletion of pKT021 [9] If LolB function is expressed from the transformed plasmid, the strain will grow at

42C 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

A660

0

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

PBAD, 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.

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

10 –1

10 0

10 1

10 2

10 3

10 –2

Time (h)

A660

mLolB LolB

0

0.0002 0.0005 0.001 0.002 0.005 0.01 0.02

LolB

0 0.0002 0.0005 0.001 0.002 0.005 0.01 0.02

0

1

2

3

4

5

LolB

mLolB

Amount (ng)

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.

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).35S-Labelled Lpp was detected only in the outer membrane of cells grown with LolB (left panel) In marked contrast, an appreciable portion of 35S-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, 35S-labelled Lpp released with LolA from spheroplasts was isolated and incubated at 30C 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

35S-labelled Lpp Essentially all 35S-labelled Lpp remained in the supernatants when mLolB was not added The amount of 35S-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–[35S]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 abspres-ence 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

IM OM IM

OM

OmpA

SecG

Lpp

IM OM

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, S]-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).

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 0 0.5 1.0 2.0 2.0 mLolB (ng·mL –1 )

OM (ΔLolB) IM

ppt

sup

Membrane

None

0

20

40

60

80

100

Time (min)

Pal

p s p s p s p s p s p s p s p s p s

Input

+

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–[35S]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%.

p s p s p s p s p s p s p s

PG PG/PE

p s p s p s p s p s p s p s

Time (min) CL

CL/PE

A

B

mLolB

p s

p s

0 20 40 60 80

p s

p s

PG CL 100

0

50 25 75 PG

PE

p s

CL

– +

25

100 PG

CL

PG PE

25

CL PE

Time (min)

0 20 40 60 80 100

PG/PE CL/PE

CL

PG

Fig 6 PE stimulates the mLolB-dependent membrane incorpora-tion of lipoproteins (A) The incorporaincorpora-tion 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.

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 30C 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) 35S-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

Vector LolA mLolB LolB

A660

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 of35S-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.

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 funcincorpora-tion 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

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

CA, USA) was used to purify hexahistidine-tagged proteins Antibodies against LolA and Lpp were raised in rabbits as

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

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

followed by monitoring the absorbance at 660 nm

Plasmids

To construct pYKT122 carrying lolB under the control of

inserted into the same sites of pMAN885EH [12] To

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

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

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

the culture absorbance reached 0.8, the cells were labelled

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

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

anion-exchange MonoQ column (GE Healthcare)

developed with a linear gradient of NaCl (0–1 m)

Release of lipoproteins from spheroplasts MC4100 cells were grown on M63 (NaCl)-maltose

all amino acids except methionine and cysteine The cells were converted into spheroplasts and then labelled with

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

was analysed by SDS–PAGE and fluorography

mLolB-dependent membrane incorporation of lipoproteins

supernatant after the lipoprotein release assay, as described above, was adsorbed to TALON affinity resin and eluted

of mLolB The reaction mixture was transferred to ice 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

followed by fluorography with Enlightning (NEN Life Sci-ence Products, Inc., Boston, MA, USA) To determine the

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)

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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