Abstract
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.
Highlights
The localization of proteins to different cellular and extracellular compartments has been studied intensively because of its fundamental importance to all living cells
The bacterial ATP binding cassette (ABC) export system is exemplified by the secretion of ␣-hemolysin from E. coli [18, 19]
colicin V (ColV) is synthesized as a 103-amino acid primary translation product which does not contain a typical Sec-dependent amino-terminal signal peptide, even though its aminoterminal 15 residues are processed concomitant with extracellular secretion [32, 33]
Summary
The localization of proteins to different cellular and extracellular compartments has been studied intensively because of its fundamental importance to all living cells. The results show that the processing of ColV-1 in vitro requires the intact CvaA, CvaB, and TolC transporters in the membranes.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have