Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase

The VirB11 ATPase is a putative component of the transport machinery responsible for directing the export of nucleoprotein particles (T complexes) across the Agrobacterium tumefaciens envelope to susceptible plant cells. Fractionation and membrane treatment studies showed that similar to 30% of VirB11 partitioned as soluble protein, whereas the remaining protein was only partially solubilized with urea from cytoplasmic membranes of wild-type strain A348 as well as a Ti-plasmidless strain expressing virB11 from an IncP replicon. Mutations in virB11 affecting protein function were mapped near the amino terminus (Q6L, P13L, and E25G), just upstream of a region encoding a Walker A nucleotide-binding site (F154H;L155M), and within the Walker A motif (P170L, K175Q, and Delta GKT174-176). The K175Q and Delta GKT174-176 mutant proteins partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. The virB11F154H;L155M allele was transdominant over wild-type virB11 in a merodiploid assay, providing strong evidence that at least one form of VirB11 functions as a home- or heteromultimer. An allele with a deletion of the first half of the gene, virB11 Delta 1-156, was transdominant in a merodiploid assay, indicating that the C-terminal half of VirB11 contains a protein interaction domain. Products of both virB11 Delta 1-156 and virB11 Delta 158-343, which synthesizes the N-terminal half of VirB11, associated tightly with the A. tumefaciens membrane, suggesting that both halves of VirB11 contain membrane interaction determinants.