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Volume 271, Number 45,
Issue of November 8, 1996
pp. 28057-28063
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing of Colicin V-1, a Secretable Marker Protein of a
Bacterial ATP Binding Cassette Export System, Requires Membrane
Integrity, Energy, and Cytosolic Factors
(Received for publication, June 7, 1996, and in revised form, August 7, 1996)
Xiaotian
Zhong
,
Roberto
Kolter
§
and
Phang C.
Tai
From the Department of Biology, Georgia State
University, Atlanta, Georgia 30303 and § Department of
Microbiology and Molecular Genetics, Harvard Medical School,
Boston, Massachusetts 02115
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.

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Copyright © 1996 by the American Society for Biochemistry and Molecular Biology.
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