The transition from closed to open conformation of Treponema pallidum outer membrane-associated lipoprotein TP0453 involves membrane sensing and integration by two amphipathic helices

The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 and its physiological role in Tp OM biogenesis, we solved its crystal structure and used mutagenesis to identify membrane insertion elements. The crystal structure of TP0453 consists of an α/β/α-fold and includes five stably folded AHs. In high concentrations of detergent, TP0453 transitions from a closed to open conformation by lateral movement of two groups of AHs, exposing a large hydrophobic cavity. Triton X-114 phase partitioning, liposome floatation assay, and bis-1-anilino-8-naphthalenesulfonate binding revealed that two adjacent AHs are critical for membrane sensing/integration. Using terbium-dipicolinic acid complex-loaded large unilamellar vesicles, we found that TP0453 increased efflux of fluorophore only at acidic pH. Gel filtration and cross-linking experiments demonstrated that one AH critical for membrane sensing/insertion also forms a dimeric interface. Based on structural dynamics and comparison with Mycobacterium tuberculosis lipoproteins LprG and LppX, we propose that TP0453 functions as a carrier of lipids, glycolipids, and/or derivatives during OM biogenesis.     

The ppm Operon Is Essential for Acylation and Glycosylation of Lipoproteins in Corynebacterium glutamicum

Background

Due to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), cleaved off their signal peptides by lipoprotein signal peptidase (Lsp) and, in Gram-negative bacteria, further triacylated by lipoprotein N-acyl transferase (Lnt). The existence of an active apolipoprotein N-acyltransferase (Ms-Ppm2) involved in the N-acylation of LppX was recently reported in M. smegmatis. Ms-Ppm2 is part of the ppm operon in which Ppm1, a polyprenol-monophosphomannose synthase, has been shown to be essential in lipoglycans synthesis but whose function in lipoprotein biosynthesis is completely unknown.

Results

In order to clarify the role of the ppm operon in lipoprotein biosynthesis, we investigated the post-translational modifications of two model lipoproteins (AmyE and LppX) in C. glutamicum Δppm1 and Δppm2 mutants. Our results show that both proteins are anchored into the membrane and that their N-termini are N-acylated by Cg-Ppm2. The acylated N-terminal peptide of LppX was also found to be modified by hexose moieties. This O-glycosylation is localized in the N-terminal peptide of LppX and disappeared in the Δppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX O-glycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed.

Conclusion

Together, these results show for the first time that Cg-Ppm1 (Ppm synthase) and Cg-Ppm2 (Lnt) operate in a common biosynthetic pathway in which lipoprotein N-acylation and glycosylation are tightly coupled.     

Functional analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative secretome analysis of a mycobacterial lgt mutant

Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [(14)C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target.     

Roles of the hydrophobic cavity and lid of LolA in the lipoprotein transfer reaction in Escherichia coli

LolA, a periplasmic chaperone, binds to outer membrane-specific lipoproteins released from the inner membrane through the action of an ATP-binding cassette transporter, LolCDE and then transfers them to the outer membrane receptor LolB, thereby mediating the inner to outer membrane transport of lipoproteins. The crystal structure of free LolA revealed that it has an internal hydrophobic cavity, which is surrounded by hydrophobic residues and closed by a lid comprising alpha-helices. The hydrophobic cavity most likely represents the binding site for the lipid moiety of a lipoprotein. It is speculated that the lid undergoes opening and closing upon the binding and transfer of lipoproteins, respectively. To determine the functions of the hydrophobic cavity and lid in detail, 14 residues involved in the formation of these structures were subjected to random mutagenesis. Among the obtained 21 LolA derivatives that did not support growth, 14 were active as to the binding of lipoproteins but defective in the transfer of lipoproteins to LolB, causing the periplasmic accumulation of a lipoprotein as a complex with a LolA derivative. A LolA derivative, I93G, bound lipoproteins faster than wild-type LolA did, whereas it did not transfer associated lipoproteins to LolB. When I93G and wild type LolA co-existed, lipoproteins were bound only to I93G; which therefore exhibited a dominant negative property. Another derivative, L59R, was also defective in the transfer of lipoproteins to LolB but did not exhibit a dominant negative property. Taken together, these results indicate that both the hydrophobic cavity and the lid are critically important for not only the binding of lipoproteins but also their transfer.     

Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae

Chimeras created by fusing the monomeric red fluorescent protein (RFP) to a bacterial lipoprotein signal peptide (lipoRFPs) were visualized in the cell envelope by epifluorescence microscopy. Plasmolysis of the bacteria separated the inner and outer membranes, allowing the specific subcellular localization of lipoRFPs to be determined in situ. When equipped with the canonical inner membrane lipoprotein retention signal CDSR, lipoRFP was located in the inner membrane in Escherichia coli, whereas the outer membrane sorting signal CSSR caused lipoRFP to localize to the outer membrane. CFSR-RFP was also routed to the outer membrane, but CFNSR-RFP was located in the inner membrane, consistent with previous data showing that this sequence functions as an inner membrane retention signal. These four lipoproteins exhibited identical localization patterns in a panel of members of the family Enterobacteriaceae, showing that the lipoprotein sorting rules are conserved in these bacteria and validating the use of E. coli as a model system. Although most predicted inner membrane lipoproteins in these bacteria have an aspartate residue after the fatty acylated N-terminal cysteine residue, alternative signals such as CFN can and probably do function in parallel, as indicated by the existence of putative inner membrane lipoproteins with this sequence at their N termini.     

Lipoprotein trafficking in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

Subcellular localisation of lipoproteins of <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> by the identification of outer membrane vesicles components

Vibrio vulnificus, a Gram-negative halophilic bacterium, is an opportunistic human pathogen that is responsible for the majority of seafood-associated deaths worldwide. Lipoproteins are important components of the bacterial cell envelope and have been shown to be involved in a wide variety of cellular processes. Little is known about the localisation or transport mechanism of lipoproteins in V. vulnificus. To assess the localisation of lipoproteins in V. vulnificus, we tested two established techniques for the rapid separation of membrane-associated proteins: detergent extraction with Sarkosyl and outer membrane vesicles (OMVs) preparation. The results showed that Sarkosyl extraction was not useful for the separation of lipoproteins from the different membranes of V. vulnificus. On the other hand, we confirmed that OMVs produced by V. vulnificus contained lipoproteins from the outer but not the inner membrane. Analysis of the OMVs components confirmed the localisation of several well-known lipoproteins to membranes that were different from expected, based on their predicted functions. Using this technique, we found that Asp at position +2 of mature lipoproteins can function as an inner membrane retention signal in V. vulnificus. Interestingly, the Escherichia coli “+2 rule” does not apply to the V. vulnificus lipoprotein IlpA (G2D) mutant, as a Ser to Asp mutation at position +2 of IlpA did not affect its outer membrane localisation. Furthermore, an IlpA tether-mRFP chimeric lipoprotein and its G2D mutant also behaved like IlpA. Together, these results suggest that the sorting rule of lipoproteins in V. vulnificus might be different from that in E. coli.     

Thematic Review Series: Proteomics: Lipoproteomics: using mass spectrometry-based proteomics to explore the assembly, structure, and function of lipoproteins

Lipoproteins are centrally important in lipid transport, fuel metabolism, and cardiovascular disease. The prototypic lipoprotein has an outer shell of amphipathic lipids and proteins that solubilizes a hydrophobic lipid core. Lipoprotein-associated proteins have classically been viewed as structural elements and factors important in lipid metabolism. Recent mass spectrometric analyses reveal that the protein cargo of lipoproteins is much more diverse than previously appreciated, raising the possibility that lipoproteins play previously unsuspected roles in host defense mechanisms and inflammation. They further suggest that lipoprotein-associated proteins can identify humans at increased risk of cardiovascular disease. Here, we summarize recent developments in lipoproteomics, the proteomic analysis of lipoproteins. We also discuss the promises and challenges this powerful analytical strategy offers for expanding our understanding of the biology and structures of lipoproteins.     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

Amino acids at positions 3 and 4 determine the membrane specificity of Pseudomonas aeruginosa lipoproteins

Escherichia coli lipoproteins with Asp at position 2 remain in the inner membrane, whereas those having other amino acids are targeted to the outer membrane by the Lol system. However, inner membrane lipoproteins without Asp at position 2 are found in other Gram-negative bacteria. MexA of Pseudomonas aeruginosa, an inner membrane-specific lipoprotein involved in multidrug efflux, has Gly at position 2. To identify the residue or region of MexA that functions as an inner membrane retention signal, we constructed chimeric lipoproteins comprising various regions of MexA and an outer membrane lipoprotein, OprM, and analyzed their membrane localization. Lys and Ser at positions 3 and 4, respectively, were found to be critical for the inner membrane localization of MexA in P. aeruginosa. Substitution of these residues with Leu and Ile, which are present in OprM, was sufficient to target the chimeric lipoprotein to the outer membrane and to abolish the ability of MexA to confer drug resistance. The membrane specificity of a model lipoprotein, lipoMalE, a lipidated variant of the periplasmic maltose-binding protein of E. coli, was also determined by the residues at positions 3 and 4 in P. aeruginosa. In contrast to the widely accepted "+2 rule" for E. coli lipoproteins, these results suggest a new "+3, +4 rule" for lipoprotein sorting in P. aeruginosa, namely, the final destination of lipoproteins is determined by the residues at positions 3 and 4.     

Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli.

The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA. Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent. This LolA-independent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient. The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.     

Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.     

Importance of apolipoproteins in lipid metabolism

Lipids, which serve as a source of energy and are an important constituent of cell membrane structure, are readily stored in the body. By definition they are insoluble in water. Specific proteins called apolipoproteins interact with lipids to form soluble lipid-protein complexes called lipoproteins. It is in this form that the major lipids — cholesterol, triglyceride and phospholipid — circulate in plasma. Unesterified fatty acids, another major lipid group, are bound to albumin in the circulation. The plasma lipoproteins are complex macromolecules composed of lipids, apolipoproteins and carbohydrates. The relative proportions of these components differ markedly between lipoprotein classes.

Hyperlipidemia is a term used for increased concentrations of plasma cholesterol and/or triglycerides. Any one plasma lipid is present in several types of lipoproteins. Thus, hyperlipidemia implies the presence of hyperlipoproteinemia. The latter has important therapeutic implications. Most of the recent attempts at classification have been directed at the lipoprotein level of plasma lipid organization.

Decreased concentrations of lipids in plasma can be achieved by altering the rates of metabolism of lipoproteins. Decrease in lipoprotein synthesis, increased catabolism or impaired release from cells into the blood stream may all result in a decrease of plasma lipids. Drugs which affect one or more of these factors are used to treat hyperlipoproteinemia. In order to elucidate the mechanism of action of hypolipidemic drugs it is necessary to understand the lipoprotein defect at the molecular level. This requires a more detailed knowledge of lipoprotein metabolism than is presently available for most of the hyperlipoproteinemias.

This paper will review some of the generally accepted properties of the plasma lipoproteins, describe some difficulties which hamper the understanding of lipoprotein metabolism, and identify possible mechanisms by which drugs may affect lipoprotein metabolism.     

Characterization of six lipoproteins in the sigmaE regulon

In Escherichia coli, sigma(E) regulon functions are required for envelope homeostasis during stress and are essential for viability under all growth conditions. The E. coli genome encodes approximately 100 lipoproteins, and 6 of these are regulated by sigma(E). Phenotypes associated with deletion of each of these lipoproteins are the subject of this report. One lipoprotein, YfiO, is essential for cellular viability. However, overexpression of this protein is not sufficient to alleviate the requirement of sigma(E) for viability, suggesting that the sigma(E) regulon provides more than one essential function. The remaining five lipoproteins in the sigma(E) regulon are nonessential; cells are viable even when all five are removed simultaneously. Deletion of three nonessential lipoprotein genes (nlpB, yraP, ygfL) results in the exhibition of phenotypes that suggest they are important for maintenance of the integrity of the cell envelope. deltanlpB cells are selectively sensitive to rifampin; deltayraP cells are selectively sensitive to sodium dodecyl sulfate. Such selective sensitivity has not been previously reported. Both deltayraP and deltanlpB are synthetically lethal with surA::Cm, which encodes a periplasmic chaperone and PPIase, suggesting that NlpB and YraP play roles in a periplasmic folding pathway that functions in parallel with that of SurA. Finally, the deltayfgL mutant exhibits a broad range of envelope defects, including sensitivity to several membrane-impermeable agents, an altered outer membrane protein profile, synthetic lethality with both surA::Cm and deltafkpA::Cm strains, and sensitivity to a bactericidal permeability-increasing peptide. We suggest that this lipoprotein performs a very important but as-yet-unknown function in maintaining the integrity of the cell envelope.     

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.     

The crystal structure of the Escherichia coli lipocalin Blc suggests a possible role in phospholipid binding

Lipocalins form a large multifunctional family of small proteins (15-25 kDa) first discovered in eukaryotes. More recently, several types of bacterial lipocalins have been reported, among which Blc from Escherichia coli is an outer membrane lipoprotein. As part of our structural genomics effort on proteins from E. coli, we have expressed, crystallized and solved the structure of Blc at 1.8 A resolution using remote SAD with xenon. The structure of Blc, the first of a bacterial lipocalin, exhibits a classical fold formed by a beta-barrel and a alpha-helix similar to that of the moth bilin binding protein. Its empty and open cavity, however, is too narrow to accommodate bilin, while the alkyl chains of two fatty acids or of a phospholipid could be readily modeled inside the cavity. Blc was reported to be expressed under stress conditions such as starvation or high osmolarity, during which the cell envelope suffers and requires maintenance. These data, together with our structural interpretation, suggest a role for Blc in storage or transport of lipids necessary for membrane repair or maintenance.     

Disruption of lolCDE, encoding an ATP-binding cassette transporter, is lethal for Escherichia coli and prevents release of lipoproteins from the inner membrane

ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release. To address these issues, a chromosomal lolC-lolD-lolE null mutant harboring a helper plasmid that carries the lolCDE genes and a temperature-sensitive replicon was constructed. The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE. In addition to functional LolD, both LolC and LolE were required for growth. At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane. Furthermore, both LolC and LolE were essential for the release of lipoproteins. On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein. From these results, we conclude that the LolCDE complex is an essential ABC transporter for E. coli and the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane.     

Effects of lipoprotein overproduction on the induction of DegP (HtrA) involved in quality control in the Escherichia coli periplasm

Recent biochemical examination has revealed the presence of at least 90 different lipoproteins in Escherichia coli. Among previously identified lipoproteins, only an outer membrane lipoprotein, NlpE, is known to induce expression of the degP gene upon its overproduction. The degP gene encodes a periplasmic protease, which is thought to be involved in the digestion of unfolded proteins, and is essential for growth at high temperatures. However, it is not completely clear why NlpE overproduction causes degP expression. Moreover, among newly confirmed lipoproteins, there may be others that also induce degP expression. Therefore, we overproduced each of the 90 lipoproteins and examined the level of degP expression as beta-galactosidase activity by using a degP promoter-lacZ fusion. The extent of degP expression caused by NlpE overproduction was dependent on the mode of degP-lacZ fusion. On the other hand, new inner membrane lipoprotein YafY strongly induced degP expression irrespective of the mode of fusion even though the level of overproduced YafY was lower than that of NlpE. The induction of degP expression by YafY overproduction was dependent on the Cpx two-component system. Alteration of the lipoprotein-sorting signals of NlpE and YafY did not abolish the degP induction. However, a YafY derivative possessing the outer membrane signal remained on inner membranes. The non-lipidated derivative of NlpE did not induce degP expression, indicating that membrane anchoring is essential for degP induction. The amino acid sequences of YafY and YfjS, another inner membrane lipoprotein, are highly identical, but overproduction of the latter did not induce degP expression. Construction of various YafY-YfjS chimeric lipoproteins revealed that only a few residues located in the N- and C-terminal regions were important for the induction of DegP.