A lipoprotein signal peptide plus a cysteine residue at the amino-terminal end of the periplasmic protein beta-lactamase is sufficient for its lipid modification, processing and membrane localization in Escherichia coli.
By genetic exchange and in vitro mutagenesis a hybrid beta-lactamase was constructed that contained the pCloDF13-encoded bacteriocin release protein signal peptide plus a cysteine residue coupled to the mature portion of beta-lactamase. Immunoblotting, labelling with [3H]palmitate in the presence and absence of globomycin, and pulse-chase experiments revealed that this hybrid construct is modified with lipid and processed into a lipid-modified beta-lactamase. Subcellular localization studies revealed that this hybrid is localized both in the cytoplasmic and outer membranes of Escherichia coli cells. A mutant derivative with an incomplete lipobox (LVG instead of LVAC+1) was not processed and was found in the cytoplasmic membranes.
Department of Molecular Microbiology, Faculty of Biology, Free University, Amsterdam, The Netherlands.
B
B
Oudega
D
D
Clark
F
F
Stegehuis
M J
MJ
Majoor
J
J
Luirink
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D003545:Cysteine; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008074:Lipoproteins / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D010169:Palmitic Acids; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C052368:cloacin DF13-encoded bacteriocin release protein, E coli; D019308:Palmitic Acid; C017339:globomycin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D001618:beta-Lactamases; D003545:Cysteine