Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides

Narita, Shin‐ichiro; Tokuda, Harukuni

doi:10.1016/j.febslet.2009.05.051

Cited by 123 publications

(135 citation statements)

References 24 publications

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“…LptE is anchored to the inner leaflet of the OM via an N-terminal lipid moiety. To understand the role of this lipid anchor in bringing the LptD/E complex together in vivo, we engineered a plasmid in which the coding sequencing of LptE, excluding its signal sequence and the N-terminal lipidated cysteine (a.a. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], is placed after the sequence encoding the pelB leader peptide. This plasmid constitutively expresses a soluble version of LptE in the periplasm that is no longer lipidated at the N terminus.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

184

247

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Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1∶1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM. T he OM of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed almost entirely of LPS and the inner leaflet of phospholipids (PL) (Fig. 1) (1, 2). Building and maintaining this asymmetric bilayer is a challenge for the cell because the OM is located outside the cytoplasm, in an environment that lacks an obvious energy source such as ATP. LPS and PL are synthesized at the cytoplasmic face of the IM whereas lipoproteins and integral membrane proteins are synthesized in the cytoplasm; all these OM components must be transported across the IM and the aqueous periplasmic compartment to be assembled into the OM (3, 4). In the case of LPS assembly, the molecule must ultimately reach the outer leaflet of the OM (Fig.

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Section: Resultsmentioning

confidence: 99%

“…1). Current models propose that another ABC transporter consisting of LptB, LptC, LptF, and LptG (formerly YhbG, YrbK, YjgP, and YjgQ, respectively) extracts LPS from the IM, initiating the transport and assembly of LPS to its final destination (9)(10)(11)(12). Because LPS is amphipathic, it is unlikely to diffuse across the periplasm unassisted.…”

mentioning

confidence: 99%

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

184

247

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“…The location of at least one Lpt system component in every cellular compartment points to a coordinated and defined route enabling nascent LPS to traverse the periplasm before being translocated across the OM [15]. Although it has been known for some time that the cytoplasmic protein LptB belongs to the ABC transporter superfamily [13], the function of the IM transmembrane subunits LptF and LptG was only recently identified and they were shown to form the stable complex LptBFG in a subunit ratio of 2:1:1 [11]. This constitutes the four domains of a bacterial ABC transporter: two membranespanning domains, each with six transmembrane helices predicted to form an antiparallel heterodimer [5], and two nucleotide binding domains.…”

Section: Introductionmentioning

confidence: 99%

Characterization of interactions between LPS transport proteins of the Lpt system

Bowyer

Baardsnes

Ajamian

et al. 2011

Biochemical and Biophysical Research Communications

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The lipopolysaccharide transport system (Lpt) in Gram-negative bacteria is responsible for transporting lipopolysaccharide (LPS) from the cytoplasmic surface of the inner membrane, where it is assembled, across the inner membrane, periplasm and outer membrane, to the surface where it is then inserted in the outer leaflet of the asymmetric lipid bilayer. The Lpt system consists of seven known LPS transport proteins (LptA-G) spanning from the cytoplasm to the cell surface. We have shown that the periplasmic component, LptA is able to form a stable complex with the inner membrane anchored LptC but does not interact with the outer membrane anchored LptE. This suggests that the LptC component of the LptBFGC complex may act as a dock for LptA, allowing it to bind LPS after it has been assembled at the inner membrane. That no interaction between LptA and LptE has been observed supports the theory that LptA binds LptD in the LptDE homodimeric complex at the outer membrane.Crown

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“…As LptA was shown to bind lipid A (15), it might act as a chaperone, assisting the amphipathic LPS molecules to pass through the aqueous periplasm. Furthermore, the inner membrane proteins LptB, LptC, LptF, and LptG, which form a complex (16), were also implicated in LPS transport because of their essential nature and because their depletion resulted in a phenotype similar to that of strains depleted for LptD or LptE (13,17). Furthermore, LptC was shown to bind LPS (18).…”

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confidence: 99%

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis

Bos

Tommassen

2011

Journal of Biological Chemistry

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The biosynthesis of lipopolysaccharide (LPS) in Gram-negative bacteria is well understood, in contrast to the transport to its destination, the outer leaflet of the outer membrane. In Escherichia coli, synthesis and transport of LPS are essential processes. Neisseria meningitidis, conversely, can survive without LPS and tolerates inactivation of genes involved in LPS synthesis and transport. Here, we analyzed whether the LptA, LptB, LptC, LptE, LptF, and LptG proteins, recently implicated in LPS transport in E. coli, function similarly in N. meningitidis. None of the analyzed proteins was essential in N. meningitidis, consistent with their expected roles in LPS transport and additionally demonstrating that they are not required for an essential process such as phospholipid transport. As expected, the absence of most of the Lpt proteins resulted in a severe defect in LPS transport. However, the absence of LptE did not disturb transport of LPS to the cell surface. LptE was found to be associated with LptD, and its absence affected total levels of LptD, suggesting a chaperone-like role for LptE in LptD biogenesis. The absence of a direct role of LptE in LPS transport was substantiated by bioinformatic analyses showing a low conservation of LptE in LPSproducing bacteria. Apparently, the role of LptE in N. meningitidis deviates from that in E. coli, suggesting that the Lpt system does not function in a completely conserved manner in all Gram-negative bacteria.Lipopolysaccharide (LPS) is a glycolipid, uniquely present at the cell surface of Gram-negative bacteria. It consists of a hydrophobic membrane anchor, called lipid A, which is embedded in the outer membrane (OM), 2 and an extracellular oligosaccharide core extended in some bacteria with a polysaccharide moiety, the O-antigen. The lipid A part can evoke strong, often toxic, innate immune responses; hence, it is also known as endotoxin. LPS is an essential component of the OM of most Gram-negative bacteria. Therefore, molecules involved in its biogenesis may represent attractive antimicrobial targets, as demonstrated by the successful development of antibiotics targeting LpxC, an enzyme involved LPS biogenesis (1), and LptD, an OM protein involved in LPS transport (2). However, much of the LPS biogenesis is not understood yet. The complete lipid A biosynthetic pathway, which takes place in the cytoplasm and at the inner leaflet of the inner membrane, has been elucidated in Escherichia coli and Salmonella. The ubiquitous presence of the lipid A biosynthetic enzymes in most other Gram-negative organisms suggests that this process is well conserved (3). The transport process of LPS from its site of synthesis to the cell surface is much less well understood (4 -7).The ATP-binding cassette transporter MsbA is thought to transport the lipid A-core moiety of newly synthesized LPS over the inner membrane (8, 9). At the OM, two proteins have been shown to be involved in the transport of LPS to the cell surface: the integral OM protein designated LptD (formerly Imp/OstA) (...

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Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides

Cited by 123 publications

References 24 publications

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Characterization of interactions between LPS transport proteins of the Lpt system

The LptD Chaperone LptE Is Not Directly Involved in Lipopolysaccharide Transport in Neisseria meningitidis

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