Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by the Yersinia pseudotuberculosis YopB protein.
YopB is a 401-amino-acid protein that is secreted by a plasmid-encoded type III secretion system in pathogenic Yersinia species. YopB is required for Yersinia spp. to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce apoptosis. YopB contains two predicted transmembrane helices (residues 166 to 188 and 228 to 250) that are thought to insert into the host plasma membrane during translocation. YopB is also required for pore formation and host-cell-signaling responses to the type III machinery, and these functions of YopB may also require membrane insertion. To elucidate the importance of membrane insertion for YopB function, YopB proteins containing helix-disrupting double consecutive proline substitutions in the center of each transmembrane domain were constructed. Yersinia pseudotuberculosis strains expressing the mutant YopB proteins were used to infect macrophages or epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of helix-disrupting substitutions into the second transmembrane domain of YopB resulted in a nonfunctional protein that was not secreted by the type III machinery. Introduction of helix-disrupting substitutions into the first transmembrane domain of YopB resulted in a protein that was fully functional for secretion and for interaction with YopD, another component of the translocation machinery. However, the YopB protein with helix-disrupting substitutions in the first transmembrane domain was partially defective for translocation, pore formation, and signaling, suggesting that all three functions of YopB involve insertion into host membrane.
Ryndak
Michelle B
MB
Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, 130 Life Sciences, SUNY at Stony Brook, Stony Brook, NY 11794-5222, USA.
Chung
Hachung
H
London
Erwin
E
Bliska
James B
JB
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry; D051379:Mice; D017433:Protein Structure, Secondary; D021381:Protein Transport; D015398:Signal Transduction; D015011:Yersinia pseudotuberculosis / Q000502:physiology*
D001425:Bacterial Outer Membrane Proteins; C078909:YopB protein, Yersinia; C078910:YopD protein, Yersinia; C063239:yopE protein, Yersinia