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

Abstract

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.

Effects of Lipoprotein Overproduction on the Induction of DegP (HtrA) Involved in Quality Control in the Escherichia coli Periplasm

“…The LolCDE complex, an ATP binding cassette transporter, releases outer membrane-directed lipoproteins from the inner membrane (4,5) leading to the formation of a water-soluble complex comprising one molecule each of a lipoprotein and LolA in the periplasm (6,7). The LolA-lipoprotein complex then interacts with the outer membrane receptor LolB, which catalyzes the anchoring of lipoproteins to the outer membrane (8,9).…”

Characterization of the Pseudomonas aeruginosa Lol System as a Lipoprotein Sorting Mechanism

“…These plasmids were introduced into E. coli JM83, and then expression of PaLolC, PaLolD-His, and PaLolE was induced by the addition of arabinose and IPTG. PaLolC and PaLolE were copurified with His-tagged PaLolD by metal affinity chromatography from membranes solubilized with DDM, indicating that these proteins form a complex as in the case of the E. coli LolCDE complex (25). Interestingly, the subunit composition of the purified complex varied with the concentration of IPTG that was added to induce PaLolE (Fig.…”

“…The mixture was diluted with 900 l of 50 mM Tris-HCl (pH 7.5) containing 5 mM MgSO 4 and 100 mM NaCl and then dialyzed against 2,000 ml of the same buffer at 4°C overnight. Reconstituted proteoliposomes were collected by centrifugation at 100,000 ϫ g for 2 h at 4°C and then subjected to the lipoprotein release assay at 30°C for 20 min in the presence of 4 g of LolA and 2 mM ATP as reported previously (25). Where specified, sodium orthovanadate was added to the reaction mixture at 5 mM.…”

“…In contrast, when 1 mM IPTG was added in the presence of 0.2% arabinose, only PaLolE was co-purified with PaLolD-His, although PaLolC was expressed, suggesting that excess PaLolE excluded PaLolC from the complex. The LolCDE complex of E. coli is composed of one molecule each of LolC and LolE and two copies of LolD (25). The PaLolCDE complex with the same subunit stoichiometry was purified from cells grown in the presence of 0.2% arabinose and 20 M IPTG (Fig.…”

“…The efficiency of lipoprotein release from proteoliposomes is generally low because the orientation of the reconstituted proteins is random, which leaves a major fraction of lipoproteins incompetent as to release (6,25). Nevertheless, reconstitution of LolCDE revealed important aspects of the lipoprotein release reaction (6,7,21).…”