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J. Biol. Chem., Vol. 276, Issue 6, 4245-4250, February 9, 2001
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From the University of Manchester, 1.800 Stopford Building, School
of Biological Sciences, Oxford Road,
Manchester, M13 9PT, United Kingdom
The cell surface expression of group 2 capsular
polysaccharides involves the translocation of the polysaccharide from
its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the
repeat structure of the polysaccharide, and translocation across the
periplasm requires the cytoplasmic membrane-anchored protein KpsE and
the periplasmic protein KpsD. In this paper we establish the topology
of the KpsE protein and demonstrate that the C terminus interacts with
the periplasmic face of the cytoplasmic membrane. By chemical
cross-linking we show that KpsE is likely to exist as a dimer and that
dimerization is independent of the other Kps proteins or the synthesis
of capsular polysaccharide. No interaction between KpsD and KpsE could
be demonstrated by chemical cross-linking, although in the presence of
both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of
unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer
membrane fractions and that the correct membrane association of KpsD
was dependent on KpsE, Lpp, and the secreted polysaccharide molecule.
Both KpsD and KpsE showed increased proteinase K sensitivity in the
different mutant backgrounds, reflecting conformational changes in the
KpsD and KpsE proteins as a result of the disruption of the transport
process. Collectively the data suggest that the trans-periplasmic
export involves KpsD acting as the link between the cytoplasmic
membrane transporter and the outer membrane with KpsE acting to
facilitate this transport process.
The Transport of Group 2 Capsular Polysaccharides across the
Periplasmic Space in Escherichia coli
ROLES FOR THE KpsE AND KpsD PROTEINS*
§,¶,
,
*
This work was supported in part by the BBSRC, the Wellcome
Trust, and the Lister Institute for Preventive Medicine.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Both authors contributed equally to this work.
§
Recipient of a Wellcome Travel fellowship.
¶
Recipient of an MRC quota studentship.
Recipient of a Royal Thailand Student'ship.
**
To whom correspondence should be addressed: 1.800 Stopford Bldg.,
School of Biological Sciences, University of Manchester, Oxford Rd.,
Manchester M13 9PT, UK. Tel.: 44 161 275 5601; Fax: 44 161 275 5656;
E-mail: ISRobert@fs1.scg.man.ac.uk.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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