PROCESSING OF COLICIN V-1, A SECRETABLE MARKER PROTEIN OF A BACTERIALATP BINDING CASSETTE EXPORT SYSTEM, REQUIRES MEMBRANE INTEGRITY, ENERGY, AND CYTOSOLIC FACTORS

Extracellular secretion of the peptide antibiotic colicin V (ColV) in Escherichia coil 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) sup erfamily. An amino-terminal export signal of ColV is specific for the CvaA-CvaE-TolC exporter and is processed concomitant with secretion. I n this study, we attempt to characterize this processing with a secret able marker protein, ColV-1, using a newly developed in vitro assay. P rocessing is found to be dependent on both CvaA-CvaB transporters and the TolC protein and to require membrane integrity. An additional cyto plasmic soluble factor(s) is also necessary for the processing. Althou gh 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-ethylmal eimide. Furthermore, the processing requires energy in the form of nuc leotide 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 factor s for rapid cleavage.