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Lookup NU author(s): Dr Paul Race,
Professor Jeremy LakeyORCiD,
Dr Mark Banfield
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Insertion of the enteropathogenic Escherichia coli Tir protein into the plasma membrane of intestinal epithelial cells is a crucial event in infection because it provides a receptor for intimate bacterial adherence. This interaction with the bacterial outer membrane protein intimin is also essential in generating a number of signaling activities associated with virulence. Tir can be modified at various sites by phosphorylation and functionally interacts with multiple host proteins. To investigate the mechanism of membrane insertion and to establish amodel system in which the multiple interactions/functions of Tir can be uncoupled and independently characterized, we used intrinsic tryptophan fluorescence, surface plasmon resonance, and protease digestion assays to show that Tir can insert directly into phospholipid vesicles in a composition-dependent manner to generate the topology reported in vivo. This is the first time that Tir has been shown to insert into membranes in a simple model system in the absence of chemical modification or other factors. These data are consistent with the protein interacting with lipids through two sites. The major site is localized to the transmembrane/intimin-binding domain region and includes Trp235, which is shown to be an effective reporter of interaction. The minor site is located within the C-terminal domain. Together, these data support a model in which Tir is released into the cytoplasm by the type III translocon and then independently inserts into the plasma membrane from a cytoplasmic location. A thorough understanding of this mechanism will be crucial to understand the subtleties of enteropathogenic E. coli pathogenesis. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Author(s): Race PR, Lakey JH, Banfield MJ
Publication type: Article
Publication status: Published
Journal: Journal of Biological Chemistry
ISSN (print): 0021-9258
ISSN (electronic): 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology, Inc.
PubMed id: 16436373
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