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: 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 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). 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