YscU cleavage and the assembly of Yersinia type III secretion machine complexes

YscU, a component of the Yersinia type III secretion machine, promotes auto-cleavage at asparagine 263 (N263). Mutants with an alanine substitution at yscU codon 263 displayed secretion defects for some substrates (LcrV, YopB and YopD); however, transport of effector proteins into host cells (YopE, YopH, YopM) continued to occur. Two yscU mutations were isolated that, unlike N263A, completely abolished type III secretion; YscU_G127D promoted auto-cleavage at N263, whereas YscU_G270N did not. When fused to glutathione S-transferase (Gst), the YscU C-terminal cytoplasmic domain promoted auto-cleavage and Gst-YscU_C also exerted a dominant-negative phenotype by blocking type III secretion. Gst-YscU_C/N263A caused a similar blockade and Gst-YscU_C/G270N reduced secretion. Gst-YscU _C and Gst-YscU_C/N263A bound YscL, the regulator of the ATPase YscN, whereas Gst-YscU_C/G270N did not. When isolated from Yersinia, Gst-YscU_C and Gst-YscU_C/N263A associated with YscK-YscL-YscQ; however, Gst-YscU_C/G270N interacted predominantly with the machine component YscO, but not with YscK-YscL-YscQ. A model is proposed whereby YscU auto-cleavage promotes interaction with YscL and recruitment of ATPase complexes that initiate type III secretion.