Abstract
The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.
Highlights
LolB accepts lipoproteins from LolA and anchors them to the inner leaflet of outer membranes
Seventeen conserved residues of mLolB were subjected to random mutagenesis in pRT102 carrying mLolB(His) under the control of Ptac followed by transformation into KT60/pYKT122
Two hundred transformants were isolated for each mutagenized residue, and their growth was examined on LB plates with or without arabinose and/or isopropyl -D-thiogalactopyranoside (IPTG)
Summary
LolB accepts lipoproteins from LolA and anchors them to the inner leaflet of outer membranes. Mature lipoproteins in Escherichia coli and other proteobacteria are sorted to the outer membrane by the Lol system, in which an ATP binding cassette transporter in the inner membrane releases outer membrane-specific lipoproteins in an ATP-dependent manner This causes the formation of a watersoluble complex between a lipoprotein and LolA, a periplasmic chaperone. The level of major outer membrane lipoprotein Lpp is about 106 molecules in a single cell [4, 5] and forms a lethal covalent linkage with the peptidoglycan when mislocalized in the inner membrane [6] Because of these properties, Lpp is unfavorable for the isolation of mutants defective in lipoprotein biogenesis and sorting. We tried to isolate mLolB mutants by random mutagenesis of 17 conserved residues other than Trp. Most derivatives were insoluble, but mutants as to leucine 68 remained largely soluble and were useful for examining the activities after purification. We show here that the loop of mLolB protruding into an aqueous environment is critical for both the receptor and membrane targeting activities
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