Biogenesis and Membrane Targeting of Lipoproteins
SHIN-ICHIRO NARITA AND HAJIME TOKUDA*
[SECTION EDITOR: TRACY PALMER]
Posted 21 October, 2010
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113–0032, Japan
*Corresponding author. Present address: Faculty of Nutritional Sciences, University of Morioka, 808 Sunagome, Takizawa, Iwate 020–0183, Japan
Phone: +81-19-688-5555, Fax: +81-19-688-5577, E-mail:
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The outer membrane of gram-negative bacteria is composed of a lipid bilayer comprising phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet (64). Two different types of proteins are localized in the outer membrane, outer membrane proteins (OMPs) and lipoproteins (Fig. 1). These proteins are synthesized in the cytoplasm and then translocated across the inner membrane to reach the outer membrane. Proteins destined for the outer membrane do not contain α-helical transmembrane stretches, because such stretches cause retention of the proteins in the inner membrane. Instead, OMPs span the outer membrane as a β-barrel structure. Membrane insertion of most OMPs occurs in a Bam complex-dependent manner (3, 72), although insertion of some secretins has been reported to be independent of BamA (10). On the other hand, lipoproteins are modified with lipids at their amino-terminal Cys and anchored to either the inner or outer membrane through the lipid moiety. Lipoproteins are involved in various envelope functions, including formation and maintenance of cell shape, biogenesis of the outer membrane, transport of a variety of molecules, signal transduction, and cell motility, although a number of lipoproteins, including even those of Escherichia coli, still have no known function.
The N-terminal structures of lipoproteins have been studied extensively by using Lpp, the major outer membrane lipoprotein of E. coli. Lpp is also called "murein lipoprotein" because it is associated with the murein (peptidoglycan) layer, or "Braun's lipoprotein," after its discoverer (7). Lpp is covalently linked to the peptidoglycan layer through a peptide linkage between its C-terminal Lys and meso-diaminopimelic acid in peptidoglycan (6). Lpp has an N-terminal glycerylcysteine containing two ester-linked fatty acids and one amide-linked fatty acid (24). N-Acyl diacylglyceryl-cysteine is the common structure of lipoproteins of gram-negative bacteria. On the other hand, most lipoproteins in gram-positive bacteria lack the amide-linked fatty acid (56, 86). In both gram-negative and gram-positive bacteria, processing of a lipoprotein precursor to the mature form occurs on the outer surface of the cytoplasmic membrane. The pioneering work by Wu and coworkers (93) revealed three distinct modification reactions (Fig. 2): (i) formation of a thioether linkage between a Cys in the N-terminal region of a prolipoprotein and diacylglycerol by phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt), which recognizes a consensus sequence, -L-(A/S)-(G/A)-C-, called a lipobox (or lipoprotein box); (ii) cleavage of a signal peptide by a lipoprotein-specific signal peptidase (LspA, also called signal peptidase II), which converts the S-lipidated Cys into the N-terminal residue of the mature protein; and (iii) amino-acylation of this Cys by apolipoprotein N-acyltransferase (Lnt, also called phospholipid:apolipoprotein transacylase).
Lipoproteins are present in both the inner and outer membranes. The amino acid residue at position +2 determines the membrane specificities of lipoproteins in E. coli and related γ-proteobacteria. Asp at position +2 functions as an inner membrane retention signal, while 19 other amino acids at position +2 direct lipoproteins to the outer membrane. This clear-cut rule for lipoprotein localization is called the "+2 rule." Although the protein moieties of lipoproteins are mostly hydrophilic, lipoproteins are hydrophobic as a whole due to the N-terminal lipid moiety. Therefore, outer membrane-directed lipoproteins must overcome the energetically unfavorable step at which they become detached from the inner membrane to reach the outer membrane via the hydrophilic periplasm. Periplasmic LolA was discovered as a molecular chaperone that escorts lipoproteins to the outer membrane through the periplasm (52). Subsequently, four other Lol factors were found to be essential for the lipoprotein sorting to the outer membrane. An ABC transporter LolCDE complex (95) in the inner membrane initiates the outer membrane sorting by releasing lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the lipid moiety of a lipoprotein in its hydrophobic cavity (65), thereby yielding a hydrophilic complex. LolB (51) is a lipoprotein anchored to the outer membrane, accepts a lipoprotein from LolA, and then incorporates it into the outer membrane (90) (Fig. 3).
In this chapter, molecular events involved in the biogenesis and outer membrane sorting of lipoproteins are described.
Lipoproteins as well as see (Chapter Bioinformatics and Systems Biology )periplasmic proteins and OMPs are synthesized in the cytoplasm as precursors with N-terminal signal peptides. The signal peptides of lipoproteins, which are cleaved by LspA, are similar to those of see (Chapter Molecular Architecture and Composition of Cells)periplasmic proteins and OMPs cleaved by signal peptidase I (SPase I). Both types of signal peptides comprise an n-region, an h-region, and a c-region. Positively charged residues are present in the n-region. The h-region is composed of stretches of hydrophobic and uncharged residues. SPase I-specific signal peptides have a polar c-region before the cleavage site, whereas the c-region of lipoprotein signal peptides contains a lipobox (38, 92). In addition, the h-region of LspA-specific signal peptides is shorter than those specific to SPase I (35). Taking advantage of the characteristics of LspA-specific signal peptides, programs such as LipoP (35) have been developed to predict lipoproteins, and a database of lipoproteins encoded by bacterial genomes (DOLOP) is available (48). LipoP predicts 101 putative lipoproteins encoded by the genome of E. coli K-12 (35). Most of them have been biochemically proven to encode lipoproteins (87). The E. coli K-12 strain thus encodes at least 90 species of lipoproteins. As the signal peptides for lipoproteins are similar to those for periplasmic proteins and OMPs, a common machinery, Sec translocon, translocates their precursor forms across the inner membrane (Fig. 2). Indeed, the precursor form of Lpp accumulates at nonpermissive temperature in secA, secD, secE, secF, and secY temperature-sensitive mutants (26, 78). Moreover, six sec genes (secA, secB, secD, secE, secF, and secY) are required for the maturation of prepullulanase of Klebsiella oxytoca expressed in E. coli (70). Physical interaction between lipoproteins with SRP, SecA, SecY, and YidC has been demonstrated by cross-linking experiments (14). The Tat (twin arginine translocation) pathway might also translocate lipoproteins. Although no Tat-dependent lipoprotein has been predicted or experimentally shown in E. coli or Salmonella enterica serovar Typhimurium, some genes are predicted to encode Tat pathway-dependent lipoproteins in both gram-positive and gram-negative bacteria (20, 46).
SPase I cleaves signal peptides when the cleavage site is translocated to the periplasmic side of the inner membrane (11). In contrast, cleavage of signal peptides by LspA requires modification of Cys in the lipobox with a diacylglyceryl moiety after translocation to the periplasmic side (Fig. 2). When LspA is inhibited by a specific inhibitor, globomycin, pro-Lpp modified with diacylglyceride accumulates in the inner membrane (32). Thus, LspA recognizes peptidyl diacylglycerylcysteine. On the other hand, it has been reported that LspA of Listeria monocytogenes (2) and several streptococcal species (9, 13, 28) can cleave nonlipidated prolipoproteins at the correct position.
Lgt is an inner membrane protein and catalyzes the transfer of the diacylglyceryl moiety from phosphatidylglycerol to the sulfhydryl group of Cys in the lipobox, resulting in the generation of diacylglyceryl prolipoprotein and sn-glycerol 1-phosphate (73). The program TMHMM (40) predicts that Lgt spans the membrane five times and exposes the C terminus to the cytoplasm. Fusion of the green fluorescent protein supports the cytoplasmic see also (Chapter Intracellular Voyeurism: Examining the Modulation of Host Cell Activities by Salmonella enterica Serovar Typhimurium) location of the C terminus (12). On the other hand, other programs, such as TMpred (30) and SOSUI (29), predict seven transmembrane segments for Lgt. Furthermore, peripheral association of Lgt with the inner leaflet of the inner membrane with no transmembrane segment has been proposed by Selvan and Sankaran (75) on the basis that the Lgt activity in inner membrane For more information, see (Chapter The Nucleoid: an Overview)vesicles was extractable with water. However, prolipoproteins are not modified unless they have been translocated across the inner membrane via Sec translocon (26), indicating that Lgt catalyzes diacylglyceryl transfer at the periplasmic leaflet of the inner membrane. The precise membrane topology of this enzyme remains to be determined.
LspA, an inner membrane protein with four transmembrane segments (57), then cleaves the amide linkage between the residue (mostly glycine or alanine) at position -1 and diacylglycerylcysteine at position +1, generating an apolipoprotein (Fig. 2). The diacylglycerylcysteine at position +1 now becomes the N terminus of the apolipoproteins, which is further N-acylated on the free α-amino group of cysteine at position +1. This reaction is catalyzed by Lnt, which is located in the inner membrane with six transmembrane segments (21, 71). Lnt is classified as a member of the carbon-nitrogen hydrolase (C-N hydrolase) family based on sequence homology (68). The Glu-Lys-Cys catalytic triad, which is conserved among members of C-N hydrolase family, was found to be essential for the Lnt activity, indicating that catalytic reactions proceed via the formation of an acyl enzyme intermediate (91). Unlike Lgt, which uses phosphatidylglycerol as the major diacylglyceryl donor, Lnt can utilize all species of major phospholipids of E. coli, i.e., phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin (23, 42). While the fatty acids of the thioether-linked diacylglyceryl moiety are similar to those of phospholipids, the amide-linked fatty acid is predominantly palmitate (24). In contrast, purified Lnt can utilize various phospholipids containing fatty acyl chains of various lengths and degrees of saturation as acyl chain donors (15). Orthologs of lnt are widespread in gram-negative bacteria but are not found in gram-positive bacteria except for actinomycetes. An lnt ortholog of Mycobacterium smegmatis was reported to catalyze the N-acylation of lipoproteins (89). It was also reported that N-acylated lipoproteins are present in Bacillus subtilis and Staphylococcus aureus (27, 41, 63), in which an Lnt ortholog is not found, suggesting that an enzyme different from Lnt is involved in the N-acylation of lipoproteins in these bacteria. N-Acylation is essential for outer membrane targeting of lipoproteins through the Lol pathway (15). The physiological role of N-acylation in gram-positive bacteria is not clear.
The lgt, lspA, and lnt genes are essential for the growth of E. coli and S. enterica serovar Typhimurium. Therefore, only conditional lethal mutants of these genes have been isolated (16, 17, 22, 71, 97). However, the temperature-sensitive phenotypes of these mutants are suppressed by the lack of the lpp gene (16, 17, 22). Since the number of Lpp molecules in a single cell is very high and can reach ~106 (8), its absence might decrease the requirement for lipoprotein-processing enzymes. Furthermore, cells lacking Lpp are rescued from the highly toxic effect of mislocalized Lpp in the inner membrane because covalent linkage between the C-terminal Lys of mislocalized Lpp and peptidoglycan is lethal to E. coli (96). It was also reported that the deletion of C-terminal Lys of Lpp partially restores growth of an lnt mutant (71). Because Lgt transfers the diacylglyceryl moiety from phosphatidylglycerol to Cys, it might be expected that phosphatidylglycerol is essential for the growth of E. coli and S. enterica serovar Typhimurium. However, this is not the case because lethality of a pgsA null mutant of E coli, which lacks phosphatidylglycerophosphate synthase and therefore cannot synthesize phosphatidylglycerol as well as cardiolipin, is suppressed by deletion of the lpp gene (37). It has been reported that lipoproteins are lipidated in cells lacking phosphatidylglycerol (79). These results indicate that phosphatidylglycerol is not the exclusive diacylglyceryl donor for prolipoproteins and that Lgt can catalyze the transfer of the diacylglyceryl moiety from phospholipids other than phosphatidylglycerol. In contrast, null mutants of lgt, lspA, and lnt have not been isolated. This is presumably because essential lipoproteins such as LolB (81), BamD (67), and LptE (94) cannot be targeted to the outer membrane if modification of their N-terminal Cys is incomplete.
SORTING SIGNALS FOR LIPOPROTEINS
Both Lpp and Pal are abundant lipoproteins found earlier in the outer membrane (7, 55). Many less abundant lipoproteins were found later in not only the outer but also the inner membrane of gram-negative bacteria (33).
Inouye and collaborators first found the importance of Asp at position +2 for the inner membrane localization of E. coli lipoproteins (98). They showed that replacement of Ser at position +2 of an outer membrane-specific lipoprotein by Asp caused the protein to remain in the inner membrane. Furthermore, replacement of Asp at position +2 of an inner membrane-specific lipoprotein by another residue caused outer membrane localization of the protein. From these results it was concluded that the amino acid residue at position +2 of lipoproteins functions as a sorting signal of lipoproteins. The +2 rule of lipoprotein sorting thus formulated was proved to be fundamentally correct by comprehensive analyses of the relationship between the species of residues at position +2 and the LolA-dependent release of lipoproteins from the inner membrane (85). However, it was found that the residue at position +3 also influences the sorting of native lipoproteins (18, 76, 85). For example, Asn at position +2 is an outer membrane signal but becomes an inner membrane signal when Asp is present at position +3, as found with a native inner membrane lipoprotein, AcrE. Moreover, Phe, Gly, Pro, Trp, and Tyr at position +2 also function as inner membrane signals when Asn is present at position +3, although these sequences are not found in native lipoproteins (76, 85). Bacteriocin release protein (BRP) is located in the outer membrane even after substitution of Gln at position +2 with Asp (19). On the contrary, a methionine-binding lipoprotein, MetQ, has Gly and Gln at positions +2 and +3, respectively, and is located in the inner membrane (53). The secondary or tertiary structures of BRP and MetQ might affect their localization. An inner membrane lipoprotein, CyoA, constitutes the cytochrome bo3 ubiquinol oxidase complex but has an outer membrane sorting signal, Asn and Ser at positions +2 and +3, respectively. The inner membrane retention of CyoA is determined by two transmembrane segments (47). The physiological meaning of lipid modification of CyoA is unknown because N-terminal Cys is dispensable for its function (47).
Although there are some exceptions, as described above, localization of red fluorescent lipoproteins revealed in vivo that the +2 rule of lipoprotein sorting is generally conserved among enterobacteria including E. coli, S. enterica serovar Typhimurium, Shigella flexneri, Yersinia pseudotuberculosis, Erwinia carotovora, and Klebsiella oxytoca (44). On the other hand, the residue at position +2 does not always determine the localization of lipoproteins in gram-negative bacteria other than enterobacteria. For example, the residues at positions +3 and +4 function as the sorting signal for lipoproteins in Pseudomonas aeruginosa (45, 60). However, since various residues are present at positions +3 and +4 of P. aeruginosa lipoproteins no matter whether they are inner membrane-specific or outer membrane-specific, it is not yet clear how the residues at positions +3 and +4 affect the localization of lipoproteins. Proteoliposomes reconstituted with LolCDE purified from E. coli and P. aeruginosa revealed that differences in the inner membrane retention signal depend on the origin of LolCDE (82).
Outer membrane-directed lipoproteins, which had been processed to mature forms through the sequential actions of Lgt, LspA, and Lnt, interact with LolCDE in the inner membrane (Fig. 3). Although the release of lipoproteins from the inner membrane requires hydrolysis of ATP by LolCDE (95), binding of a lipoprotein to LolCDE takes place prior to ATP binding (34). LolCDE is considered to recognize the N-acyl diacylglyceryl-cysteine moiety of lipoproteins (25). Amino acylation of the N-terminal diacylglyceryl cysteine is essential for the recognition by LolCDE, because LolCDE does not release apolipoproteins (15). This is why apolipoproteins accumulate in the inner membrane of a conditional lnt mutant under nonpermissive conditions (71).
LolCDE does not recognize lipoproteins with Asp at position +2, because the residue functions as a LolCDE avoidance signal (50). This is why Asp at position +2 causes the inner membrane localization of lipoproteins. The distance of the negative charge from the α-carbon of the residue at position +2 is critical for the LolCDE avoidance function (25). Therefore, Glu does not replace Asp but cysteic acid generated on oxidation of Cys does, as the negative charge distance for the latter is similar to that for Asp. On the other hand, chemical modification of the residue at position +2 of an outer membrane-specific lipoprotein does not inhibit its release by LolCDE (25), indicating that LolCDE does not recognize the residue at position +2, which had been thought to function as the lipoprotein-sorting signal. Taken together, these observations indicate that LolCDE binds lipoproteins by default unless Asp is present at position +2.
Phospholipid compositions are critically important for the LolCDE avoidance function of Asp in reconstituted proteoliposomes (25, 54). Phospholipids possessing a positive charge such as phosphatidylethanolamine and phosphatidylcholine are required for the LolCDE avoidance function of Asp in vitro, although phosphatidylcholine is not a natural phospholipid in E. coli. It is speculated that an electrostatic interaction between the negative charge of the Asp and the positive charge of phospholipids inhibits the recognition of lipoproteins by LolCDE. Phosphatidylglycerol is also important, presumably for correct functioning of LolCDE.
Lipoproteins possessing Asp at position +2 are released from the inner membrane by the LolCDE mutant having the Ala-to-Pro mutation at position 40 of LolC (61). Once released, lipoproteins with Asp at position +2 are localized to the outer membrane in LolA- and LolB-dependent manners. Therefore, the LolCDE avoidance signal functions for neither LolA nor LolB.
Binding of lipoproteins and ATP to LolCDE, and subsequent hydrolysis of ATP by LolCDE cause conformational changes of LolCDE and LolA, through which lipoproteins are irreversibly sorted to the outer membrane (88). Lipoprotein binding to LolC and/or LolE increases the affinity of LolD for ATP (34). ATP binding to LolD then decreases the strength of the hydrophobic interaction between LolCDE and lipoproteins (34, 83). ATP hydrolysis induces the transfer of lipoproteins from LolCDE to LolA, leading to the formation of a water-soluble LolA-lipoprotein complex (52). The ATPase activity of LolD is inhibited by orthovanadate, an inorganic phosphate analog, like other ABC transporters (50). However, a single cycle of lipoprotein transfer from liganded LolCDE to LolA takes place even after ATP hydrolysis is completely blocked by orthovanadate (83).
The crystal structures of LolA and LolB are very similar to each other and comprise 11 antiparallel β-strands folded into an incomplete β-barrel, and the loops containing three α-helices covering the β-barrel (80). Hydrophobic cavities formed inside the LolA and LolB barrels accommodate the acyl chains of lipoproteins (65, 66). However, the hydrophobic cavity of LolA is closed on hydrogen bonding between Arg at position 43 in the β-barrel and residues in the loops. ATP hydrolysis by LolCDE causes both the transfer of lipoproteins to LolA and opening of the hydrophobic cavity of LolA (65).
LolC and LolE exhibit 26% sequence identity and have similar membrane topologies; both proteins have four transmembrane segments and one large periplasmic loop between the first and second transmembrane segments (99). Both LolC and LolE are essential for the growth of E. coli (62), while lipoprotein-releasing activity can be detected in proteoliposomes reconstituted with LolD and LolE without LolC (36). In contrast, LolA directly interacts with LolC but not with LolE (66), suggesting that lipoproteins are transferred from LolC to LolA. These observations indicate that the two membrane subunits play different roles in the release of lipoproteins.
Lipoproteins cross the periplasmic space as a soluble complex with LolA to the outer membrane, where the complex interacts with outer membrane receptor LolB (51). Lipoproteins are then irreversibly transferred from LolA to LolB, resulting in the transient formation of a LolB-lipoprotein complex (51, 90). LolB also accommodates the acyl chains of lipoproteins in its hydrophobic cavity (66, 80).
The mode of interaction between LolA and LolB was analyzed by means of in vivo photo-cross-linking and nuclear magnetic resonance (NMR) (59, 66). Both studies revealed that LolA and LolB interact with each other by connecting the entrances of their hydrophobic cavities. Thus, the hydrophobic cavities of LolA and LolB form a tunnel through which lipoproteins are quickly transported to the LolB area. The affinity of the hydrophobic cavity for lipoproteins is significantly higher with LolB than with LolA (84), partly because of the difference in the species of hydrophobic residues located inside the cavity (80). Bulky and rigid aromatic residues are abundant in LolA, while smaller hydrophobic residues such as Leu and Ileu are abundant in LolB. Replacement of Arg at position 43 of LolA by Leu causes an open conformation of LolA (65) and makes the hydrophobic interaction between LolA and lipoproteins as strong as that between LolB and lipoproteins (84). This mutation nearly completely abolishes the lipoprotein transfer from LolA to LolB. Thus, the formation of the hydrophobic tunnel, difference in affinity for lipoproteins, and closed conformation of LolA contribute to the very efficient one-way lipoprotein transfer to LolB in the periplasm, where chemical energy sources such as ATP are not available.
LolB has three functions; it accommodates the acyl chains of lipoproteins in its hydrophobic cavity, targets them to a lipid bilayer, and incorporates the associated lipoproteins into the lipid bilayer. LolB is a lipoprotein, but its lipid anchor is dispensable for the activity, and a soluble LolB derivative, mLolB, expressed in the periplasm complements the LolB function (90). However, since mLolB does not distinguish the outer and inner membranes, transient mislocalization of Lpp in the inner membrane occurs. The crystal structure of LolB revealed that Leu at position 68 is located in the loop that protrudes into the solvent region despite its hydrophobic nature (80). Replacement of Leu at position 68 by charged residues was found to impair the lipoprotein transfer to the lipid bilayer without affecting its ability to bind lipoproteins (R. Tsurumizu, Y. Hayashi, S. Narita, and H. Tokuda, unpublished observation).
The protein moiety of Lpp folds into a trimeric helical structure comprising a parallel three-stranded coiled coil and two helix-capping motifs (77). One-third of Lpp molecules form a peptide bond between the ε-amino group of their C-terminal Lys and meso-diaminopimelate in a linear stem pentapeptide of peptidoglycans (4, 43). This linkage connects the outer membrane to the peptidoglycan and contributes to the integrity of the envelope structure (8). Because a single transport cycle of the Lol pathway transports a single molecule of lipoprotein (Fig. 3), trimerization and linkage with the peptidoglycan should take place after the incorporation of Lpp into the outer membrane. Four E. coli genes, erfK, ycfS, ynhG, and ybiS, have been determined to encode transpeptidases that catalyze the covalent linkage between Lpp and the peptidoglycan by virtue of their homology to the transpeptidase of Enterococcus faecium (49). A quadruple mutant lacking all four genes does not form the covalent linkage between Lpp and the peptidoglycan (49). Since the ybiS null mutant nearly completely lacks the covalent linkage, YbiS is likely the main transpeptidase catalyzing the cross-linking between Lpp and the peptidoglycan. Although ErfK and YcfS have intrinsic activities, their physiological functions as well as that of YnhG remain to be elucidated.
P. aeruginosa lipoprotein OprM exists as a trimer in the outer membrane. CusC is homologous to OprM and is involved in the copper/silver efflux in E. coli (58). These lipoproteins are also considered to become trimerized after they have been localized in the outer membrane through the Lol pathway. The trimeric structure of OprM (1) is similar to that of TolC (39), which comprises a β-barrel domain spanning the outer membrane and an α-barrel domain forming a cavity protruding into the periplasm. The outer membrane localization of TolC requires BamA (31), a central component of the machinery catalyzing the outer membrane incorporation of the β-barrel protein (3, 72). OprM and CusC may therefore require both the Lol pathway and the Bam machinery for localization to the outer membrane.
The pathways for the biosynthesis and transport of lipoproteins to the outer membrane of E. coli have been clarified in detail. Virtually all lipoproteins in this bacterium are translocated across the inner membrane through the Sec translocon and sequentially processed to mature forms by Lgt, LspA, and Lnt. The transport of lipoproteins from the inner membrane to the outer membrane of E. coli is generally mediated by the Lol pathway (100). Because genes for Lol proteins are conserved in other gram-negative proteobacteria, the Lol pathway is thought to be a general lipoprotein-targeting pathway. On the other hand, specialized targeting machineries mediate the outer membrane localization of some lipoproteins. For example, the type II secretion system of Klebsiella strains is composed of more than 10 proteins and mediates the transport of PulA from the outer leaflet of the inner membrane to the outer leaflet of the outer membrane (69). Borrelia spirochetes express a number of lipoproteins (5), which are targeted to the bacterial surface by default but can be localized on the periplasmic side of membranes by specific sorting signals (74). The detailed molecular mechanisms whereby lipoproteins are translocated across the outer membrane remain unknown.
We thank all present and past members of this laboratory for their contributions to this chapter. The work carried out in this laboratory has been supported by grants to H.T. from the Ministry of Education, Science, Sports and Culture of Japan.
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Module4.3.7
