Mechanical fractionation reveals structural requirements for enteropathogenic Escherichia coli Tir insertion into host membranes.
Enteropathogenic Escherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system. Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells. In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins. We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin.
Gauthier
A
A
Department of Biochemistry and Molecular Biology and Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
de Grado
M
M
Finlay
B B
BB
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001693:Biological Transport, Active; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism / Q000382:microbiology; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017830:Octoxynol; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D017830:Octoxynol