Dataset Information

Reconstitution of bacterial autotransporter assembly using purified components.

ABSTRACT: Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular ('passenger') domain and a C-terminal ? barrel ('?') domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro. Our results indicate that the membrane integration of the ? domain is the rate-limiting step in autotransporter assembly and that passenger domain translocation does not require the input of external energy. Furthermore, experiments using nanodiscs strongly suggest that autotransporter assembly is catalyzed by a single copy of the Bam complex. Finally, we describe a method to purify a highly active form of the Bam complex that should facilitate the elucidation of its function.

SUBMITTER: Roman-Hernandez G

PROVIDER: S-EPMC4174580 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reconstitution of bacterial autotransporter assembly using purified components.

Roman-Hernandez Giselle G Peterson Janine H JH Bernstein Harris D HD

eLife 20140902

Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular ('passenger') domain and a C-terminal β barrel ('β') domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotranspor ...[more]

PMID: 25182416

Similar Datasets

Project description:BACKGROUND: Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. METHODOLOGY/PRINCIPAL FINDINGS: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. CONCLUSIONS/SIGNIFICANCE: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.

Dataset Information

Reconstitution of bacterial autotransporter assembly using purified components.

Publications

Reconstitution of bacterial autotransporter assembly using purified components.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets