Interactions between type III secretion apparatus components from Yersiniapestis detected using the yeast two-hybrid system

Interactions among the Yersinia secretion (Ysc) proteins of Yersinia pestis were explored using the yeast two-hybrid system. Various pairwise combinat ions of the yscEFGHIKLN and Q genes fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast, and expression of a reporter gene encoding beta-galaclosidase was detected. Combinations of yscN and yscL, yscL and yscQ, and yscQ and yscK resulted in high levels of reporter gene activation; These results suggest that YscL interacts with bo th YscN and YscQ, and that YscQ interacts with both YscL and YscK. Three-hy brid analyses using plasmid pDELA to target a third hybrid protein to the y east nucleus was used to detect the formation of ternary protein complexes. Using the three-hybrid system, YscQ expressed from plasmid pDELA was able to bring together the YscK and YscL fusion proteins. In a similar manner, Y scL expressed from plasmid pDELA was able to bring together the YscN and Ys cQ fusion proteins. Together, these results suggest that a complex composed of YscN, YscQ, YscK and YscL is involved in the assembly and/or function o f the Y. pestis type III secretion apparatus. (C) 2000 Federation of Europe an Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.