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.
Background: LolB accepts lipoproteins from LolA and anchors them to the inner leaflet of outer membranes. Results: Membrane targeting of lipoproteins is defective in several mutants with substitutions of Leu-68 of LolB.
Conclusion:The protruding loop of LolB plays critical roles in the membrane anchoring activity. Significance: A possible mechanism for the last step of lipoprotein sorting is proposed.
a b s t r a c tLipoproteins of Escherichia coli are sorted to the outer membrane through a pathway composed of five Lol proteins. LolA transports lipoproteins released from the inner membrane by LolCDE to LolB on the outer membrane via the periplasm. Interaction between LolA and LolB was speculated to be strong when LolA binds lipoprotein. However, due to a lack of a sensitive method, the kinetics of this reaction have not been examined in detail. We report here the detection of lipoprotein transfer in real time by means of surface plasmon resonance. The kinetic parameters of lipoprotein transfer were determined with wild-type LolA and a mutant defective in it.Structured summary: MINT-7259948: mlolB (uniprotkb:P61320) binds (MI:0407) to pal (uniprotkb:P0A912) by surface plasmon resonance (MI:0107)
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