INTERMOLECULAR DISULFIDE BONDS STABILIZE VIRB7 HOMODIMERS AND VIRB7 VIRB9 HETERODIMERS DURING BIOGENESIS OF THE AGROBACTERIUM-TUMEFACIENS T-COMPLEX TRANSPORT APPARATUS/

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the sta bilization of VirB proteins during biogenesis of the putative T-comple x transport apparatus, Here, we report that stabilization of VirB7 its elf is correlated with its ability to form disulfide cross-linked homo dimers via a reactive Cys-24 residue. Three types of beta-mercaptoetha nol-dissociable complexes were visualized with VirB7 and/or a VirB7::P hoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a V irB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7 /VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer. A VirB7C24S mutant protein was immunolog ically undetectable, whereas the corresponding VirB7C24S::PhoA41 deriv ative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7. We further report tha t VirB7-dependent stabilization of VirB9 is correlated with the abilit y of these two proteins to dimerize via formation of a disulfide bridg e between reactive Cys-24 and Cys-262 residues, respectively, Two type s of dissociable complexes were visualized: (i) a 36-kDa complex corre sponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa compl ex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer, a VirB 9C262S mutant protein was immunologically undetectable, whereas the co rresponding VirB9C262S::PhoA293 derivative accumulated to detectable l evels but failed to form dissociable heterodimers with wild-type VirB7 , Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus.