Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at +2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg(2+) but very high at 10 mM Mg(2+) in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg(2+), and the release of lipoproteins with Asp at +2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at +2 at 2 mM Mg(2+). The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at +2 even at 10 mM Mg(2+), while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.     

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.     

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at + 2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg²⁺ but very high at 10 mM Mg²⁺ in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg²⁺, and the release of lipoproteins with Asp at + 2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at + 2 at 2 mM Mg²⁺. The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at + 2 even at 10 mM Mg²⁺, while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.     

An ABC transporter mediating the membrane detachment of bacterial lipoproteins depending on their sorting signals

Bacterial lipoproteins are anchored to membranes through a lipid moiety attached to the N-terminal Cys. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane and others in the inner membrane. Sorting of lipoproteins to the outer membrane requires the Lol system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the lipoprotein sorting by mediating the detachment of outer membrane-specific lipoproteins from the inner membrane. LolCDE does not recognize lipoproteins possessing Asp at position 2, which therefore remain anchored to the inner membrane. We will discuss the mechanism of LolCDE based on data obtained through in vitro experiments.     

Characterization of the Pseudomonas aeruginosa Lol system as a lipoprotein sorting mechanism

Escherichia coli lipoproteins are localized to either the inner or the outer membrane depending on the residue that is present next to the N-terminal acylated Cys. Asp at position 2 causes the retention of lipoproteins in the inner membrane. In contrast, the accompanying study (9) revealed that the residues at positions 3 and 4 determine the membrane specificity of lipoproteins in Pseudomonas aeruginosa. Since the five Lol proteins involved in the sorting of E. coli lipoproteins are conserved in P. aeruginosa, we examined whether or not the Lol proteins of P. aeruginosa are also involved in lipoprotein sorting but utilize different signals. The genes encoding LolCDE, LolA, and LolB homologues were cloned and expressed. The LolCDE homologue thus purified was reconstituted into proteoliposomes with lipoproteins. When incubated in the presence of ATP and a LolA homologue, the reconstituted LolCDE homologue released lipoproteins, leading to the formation of a LolA-lipoprotein complex. Lipoproteins were then incorporated into the outer membrane depending on a LolB homologue. As revealed in vivo, lipoproteins with Lys and Ser at positions 3 and 4, respectively, remained in proteoliposomes. On the other hand, E. coli LolCDE released lipoproteins with this signal and transferred them to LolA of not only E. coli but also P. aeruginosa. These results indicate that Lol proteins are responsible for the sorting of lipoproteins to the outer membrane of P. aeruginosa, as in the case of E. coli, but respond differently to inner membrane retention signals.     

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.     

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.     

A single amino acid determinant of the membrane localization of lipoproteins in E. coli

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.     

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane,the Periplasm and Beyond

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+ 2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation, likely through interaction with a periplasmic holding chaperone, which delivers the proteins to an outer membrane lipoprotein flippase. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Lipoprotein sorting signals evaluated as the LolA-dependent release of lipoproteins from the cytoplasmic membrane of Escherichia coli

Escherichia coli lipoproteins are anchored to either the inner or outer membrane through fatty acyl chains covalently attached to an N-terminal cysteine. Aspartate at position 2 functions to retain lipoproteins in the inner membrane, although the retention is perturbed depending on the residue at position 3. We previously revealed that LolCDE and LolA play critical roles in this lipoprotein sorting. To clarify the sorting signals, the LolA-dependent release of lipoprotein derivatives having various residues at positions 2 and 3 was examined in spheroplasts. When the residue at position 3 was serine, only aspartate at position 2 caused the retention of lipoproteins in spheroplasts. We then examined the release of derivatives having aspartate at position 2 and various residues at position 3. Strong inner membrane retention occurred with a limited number of species of residues at position 3. These residues were present at position 3 of native lipoproteins having aspartate at position 2, whereas residues that inhibited the retention were not. It was also found that a strong inner membrane retention signal having residues other than aspartate at position 2 could be formed through the combination of the residues at positions 2 and 3. These results indicate that the inner membrane localization of native lipoproteins is ensured by the use of a limited number of strong inner membrane retention signals.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Outer membrane lipoprotein biogenesis: Lol is not the end

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.     

A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.

Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys. The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form. A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified. The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20. Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence. p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins. Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro. These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane.     

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.     

Bacterial lipoproteins; biogenesis,sorting and quality control

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane.This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Depletion of apolipoprotein N-acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia coli

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position +2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with beta-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.     

The protein sequence responsible for lipoprotein membrane localization in Escherichia coli exhibits remarkable specificity

Structural information defining an N-terminal sequence required for the membrane sorting of bacterial lipoproteins has been previously garnered through the study of a hybrid outer membrane (OM) lipo-beta-lactamase (LL) (Ghrayeb and Inouye (1984) J. Biol. Chem. 259, 463-467). Introduction of an aspartate as the second residue of mature LL (D2 mutant) causes an inner membrane (IM) localization of this protein (Yamaguchi, K., Yu, F., and Inouye, M. (1988) Cell 53, 423-432). Introduction of as aspartate at the third residue of mature LL (D3) causes a weaker IM sorting signal and when present as the fourth residue (D4), normal OM sorting occurs. A positively charged residue at the second position (K2) has no effect on OM localization. Remarkably, glutamate substitution at either the second (E2) or third (E3) position does not interfere with OM sorting. Sorting of the mutant D2 LL can be partially suppressed by introduction of a positively charged histidine (D2H3) or lysine (D2K3) at residue 3 of the mature protein. These results indicate that both the negative charge of the aspartate residue and some structural feature not present in a glutamate residue are required for sorting to the IM. The suppression of IM localization of the D2H3 LL double mutant can be eliminated by growing Escherichia coli at pH 8.4 to reduce the histidine partial positive charge. This result supports the essentiality of a negative charge in IM localization and indicates that the committed step in lipoprotein sorting is made in a cellular compartment, the periplasm, at equilibrium with the external pH.     

A new ABC transporter mediating the detachment of lipid-modified proteins from membranes

Lipoproteins in Escherichia coli are anchored to the periplasmic side of either the inner or the outer membrane by a lipid moiety that is covalently attached to the amino-terminal cysteine residue. Membrane specificity depends on a sorting signal at position 2 of the lipoprotein. Lipoproteins directed to the outer membrane are released from the inner membrane in an ATP-dependent manner through the formation of a complex with LolA, a periplasmic chaperone. However, the ATPase involved in this reaction has not been identified. Here we show, using reconstituted proteoliposomes, that a new complex, LolCDE, belonging to the ATP-binding cassette (ABC) transporter family, catalyses the release of lipoproteins in LolA- and sorting-signal-dependent manners. The LolCDE complex differs mechanistically from all other ABC transporters as it is not involved in the transmembrane transport of substrates. This new mechanism is evolutionarily conserved in other gram-negative bacteria.