title: Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase.
abstract: Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.
authors: Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan. snarita@virus.kyoto-u.ac.jp
authors: Shin-ichiro
authors: S
authors: Narita
authors: Hajime
authors: H
authors: Tokuda
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000235:genetics; D000217:Acyltransferases / Q000172:deficiency / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D008074:Lipoproteins / Q000032:analysis
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C407982:LolC protein, E coli; C407983:LolD protein, E coli; C407984:LolE protein, E coli; D000217:Acyltransferases; C068902:apolipoprotein N-acyltransferase