Processing of colicin V-1, a secretable marker protein of a bacterial ATP binding cassette export system, requires membrane integrity, energy, and cytosolic factors.
Extracellular secretion of the peptide antibiotic colicin V (ColV) in Escherichia coli is mediated by a dedicated exporter system consisting of host TolC protein and the products of two specific genes, cvaA and cvaB, the latter being a member of the ATP binding cassette (ABC) superfamily. An amino-terminal export signal of ColV is specific for the CvaA-CvaB-TolC exporter and is processed concomitant with secretion. In this study, we attempt to characterize this processing with a secretable marker protein, ColV-1, using a newly developed in vitro assay. Processing is found to be dependent on both CvaA-CvaB transporters and the TolC protein and to require membrane integrity. An additional cytoplasmic soluble factor(s) is also necessary for the processing. Although the sequence of the cleavage site suggests it could be a substrate, ColV-1 cannot be processed in vitro by the purified leader peptidase I. Moreover, ColV-1 processing is inhibited by antipain and N-ethylmaleimide. Furthermore, the processing requires energy in the form of nucleotide hydrolysis. These results indicate that the processing of ColV-1 is specific and more complex than expected, requiring the CvaA-CvaB-TolC transporter intact in the membrane, energy, and cytosolic factors for rapid cleavage.
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA. biopct@panther.gsu.edu
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D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000976:Antipain / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003087:Colicins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009876:Operon; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003087:Colicins; C096447:CvaA protein, E coli; C102358:CvaB protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; D000976:Antipain; D006160:Guanosine Triphosphate; D000251:Adenosine Triphosphatases; D005033:Ethylmaleimide