An intramolecular disulphide bond reduces the efficacy of a lipoprotein plasma membrane sorting signal.
To study lipoprotein sorting in Escherichia coli, we devised a novel screen in which sensitivity or resistance to bacteriophage T5 and colicin M reflects the membrane localization of the bacteriophage T5-encoded lipoprotein Llp, which inactivates the outer membrane (OM) T5 receptor (FhuA). When processed by lipoprotein signal peptidase, Llp has a serine at position +2, immediately after the fatty acylated N-terminal cysteine. As predicted by the '+2 lipoprotein sorting rule' that determines the localization of lipoproteins in the cell envelope, Llp is located in the OM. However, contrary to expectations, when serine +2 was replaced by aspartate, the canonical plasma membrane lipoprotein retention signal, Llp was still > or =40% targeted to the OM and protected cells against colicin M and phage T5. OM association of this Llp derivative was abolished when a peptide spacer was inserted between the aspartate and the rest of Llp or when the formation of an intramolecular disulphide bond in Llp was prevented by substituting one or other of the cysteines involved. Furthermore, analysis of a MalE-Llp hybrid protein with or without a lipid moiety demonstrated that fatty acylation of Llp is essential for its OM association and for protection against colicin M and bacteriophage T5. These data suggest (i) that phage-encoded Llp uses the endogenous E. coli Lol pathway for lipoprotein sorting to the OM and (ii) that the conformation of a lipoprotein can affect its sorting within the cell envelope.
Robichon
Carine
C
Unité de Génétique Moléculaire, CNRS URA 2172, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris 15, France.
Bonhivers
Mélanie
M
Pugsley
Anthony P
AP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008074:Lipoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003087:Colicins; D004220:Disulfides; D008074:Lipoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins