Lipopolysaccharides and divalent cations are involved in the formation of an assembly-competent intermediate of outer-membrane protein PhoE of E.coli.
To identify the requirements for the biogenesis of outer-membrane proteins in Gram-negative bacteria, the sorting and assembly of the trimeric, pore-forming protein PhoE was studied in vitro. Purified lipopolysaccharide (LPS) in combination with low amounts of Triton X-100 and divalent cations induced the formation of folded monomers. LPS of deep-rough strains was far less efficient in the formation of folded monomers than wild-type LPS was. These folded monomers could be converted into heat-stable trimers upon addition of outer membranes and higher amounts of Triton X-100. Trimerization could precede the insertion step. These in vitro data suggest that the assembly in vivo proceeds sequentially by (i) formation of a folded monomer by interaction with LPS; (ii) sorting of the folded monomers to assembly sites in the outer membrane; (iii) trimerization; and (iv) insertion.
Department of Molecular Cell Biology, Utrecht University, The Netherlands.
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H
de Cock
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Tommassen
D002413:Cations, Divalent / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides / Q000378:metabolism*; D008793:Metrizamide; D017830:Octoxynol; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D018272:Porins; C082922:PhoE protein, E coli; D017830:Octoxynol; D008793:Metrizamide