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J. Bacteriol., Nov 1995, 6153-6159, Vol 177, No. 21
RC Skvirsky, S Reginald and X Shen
The antibacterial protein toxin colicin V is secreted from Escherichia coli
cells by a dedicated export system that is a member of the multicomponent
ATP-binding cassette (ABC) transporter family. At least three proteins,
CvaA, CvaB, and TolC, are required for secretion via this signal
sequence-independent pathway. In this study, the subcellular location and
transmembrane organization of membrane fusion protein CvaA were
investigated. First, a series of CvaA-alkaline phosphatase (AP) protein
fusions was constructed. Inner and outer membrane fractionations of cells
bearing these fusions indicated that CvaA is inner membrane associated. To
localize the fusion junctions, the relative activities of the fusion
proteins, i.e., the amounts of phosphatase activity normalized to the rate
of synthesis of each protein, as well as the stability of each fusion, were
determined. These results indicated that all of the fusion junctions occur
on the same side of the inner membrane. In addition, the relative
activities were compared with that of native AP, and the protease
accessibility of the AP moieties in spheroplasts and whole cells was
analyzed. The results of these experiments suggested that the fusion
junctions occur within periplasmic regions of CvA. We conclude that CvaA is
an inner membrane protein with a single transmembrane domain near its N
terminus; the large C-terminal region extends into the periplasm. This
study demonstrates the application of AP fusion analysis to elucidate the
topology of a membrane-associated protein having only a single
transmembrane domain.
Copyright © 1995, American Society for Microbiology
Topology analysis of the colicin V export protein CvaA in Escherichia coli
Department of Biology, University of Massachusetts-Boston 02125, USA.
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