INTERACTIONS OF VIRB9, VIRB10, AND VIRB11 WITH THE MEMBRANE-FRACTION OF AGROBACTERIUM-TUMEFACIENS - SOLUBILITY STUDIES PROVIDE EVIDENCE FORTIGHT ASSOCIATIONS

The eleven predicted gene products of the Agrobacterium tumefaciens vi rB operon are believed to form a transmembrane pore complex through wh ich T-DNA export occurs. The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fracti on of A. tumefaciens. Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J. E. Ward, E. M. Dale, E. W. Nester, and A. N. Binns, J. Bacteriol. 172:5200-5210, 1990). Deletion of the sequences encoding amino acids 2 2 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA tran sfer to the plant cell. Western blot (immunoblot) analysis indicated t hat the observed loss of virulence could not be attributed to a decrea se in the steady-state levels of the mutant VirB10 protein. Although t he deletion of the single transmembrane domain would be expected to pe rturb membrane association, VirB10 Delta 22-55 was found exclusively i n the membrane fraction. Urea extraction studies suggested that this m embrane localization might be the result of a peripheral membrane asso ciation; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugati on. Both wild-type VirB10 and wild-type VirB9 were only partially remo ved from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble. VirB11 was stripped from the mem branes by 6 M urea but not by a more mild salt extraction. The fractio nation patterns of VirB9, VirB10, and VirB11 were not dependent on eac h other or on VirB8 or VirD4. The observed tight associations of VirB9 , VirB10, and VirB11 with the membrane fraction support the notion tha t these proteins may exist as components of multiprotein pore complexe s, perhaps spanning both the inner and outer membranes of Agrobacteriu m cells.