RESUMO
A photo-sensitive amino acid analogue was introduced into an outer membrane lipoprotein, Pal, and then subjected to photo-crosslinking with the lipoprotein-specific ABC transporter LolCDE. Pal crosslinked to LolE but not LolC in vivo despite that both are structurally similar membrane subunits. LolCDE liganded with Pal containing the photo-sensitive amino acid analogue was isolated and subjected to in vitro photo-crosslinking. LolE was found to be the binding site for Pal. ATP binding to LolD decreased the LolE-Pal crosslinking by decreasing their hydrophobic interaction. ATP hydrolysis in the presence of LolA completely abolished the LolE-Pal crosslinking and, concomitantly, generated a new LolA-Pal crosslinked product.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Benzofenonas/química , Western Blotting , Cromatografia de Afinidade , Códon de Terminação , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/isolamento & purificação , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/química , Processos Fotoquímicos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Escherichia coli cells express at least 90 species of lipoprotein. LolB is one of the essential outer membrane lipoproteins, being involved in the last step of lipoprotein sorting. It accepts lipoproteins from a periplasmic molecular chaperone, LolA, and mediates the outer membrane anchoring of lipoproteins through a largely unknown mechanism. It has been shown previously that a LolB derivative, mLolB, lacking an N-terminal acyl chain, can bind lipoproteins. We examined how the lack of an N-terminal anchor affects the outer membrane anchoring of lipoproteins. Surprisingly, mLolB compensates for LolB function and supports E. coli growth, indicating that the N-terminal anchor is not essential for its function. Indeed, mLolB correctly localizes lipoproteins to either the inner or outer membrane depending on the sorting signal at the steady state. Furthermore, periplasmic mLolB enables the dissection of LolB function, namely lipoprotein binding, membrane targeting and lipoprotein anchoring. It mediates the transfer of lipoproteins from LolA to the outer membrane, but also the inner membrane and liposomes, indicating that mLolB exhibits no membrane preference and targets to phospholipids. Consequently, an outer membrane-specific lipoprotein is transiently mislocalized to the inner membrane when cells express only mLolB. LolB anchored to the outer membrane does not cause such mislocalization and is more active than mLolB. Phosphatidylethanolamine has been found to stimulate the mLolB-dependent membrane anchoring of lipoproteins. Taken together, these results indicate that lipoprotein binding, membrane targeting and membrane incorporation of lipoproteins are intrinsic functions of LolB.
