The transfer of oncogenic nucleoprotein complexes (T-complexes) from Agrobacterium tumefaciens to susceptible plant cells is mediated by a type IV bacterial secretion system. The VirB11 ATPase is an essential component of this membrane-associated transport system. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of the T-complex transporter. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that ~30 % of VirB11 partitioned as soluble protein and ~70% was tightly associated with the bacterial cytoplasmic membrane. Membrane treatment experiments showed that VirB11 remained tightly associated with the cytoplasmic membrane upon treatment with various chemical reagents and did not simply co-sediment with membranes as protein aggregates. No differences were detected in VirB11 membrane association in the presence or absence of other transport system components. To identify residues or domains important for protein localization and function, the virB11 sequence was mutagenized by random or site-directed mutagenesis. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker ‘A’ nucleotide-binding site, and within the Walker ‘A’ motif. The two derivatives with mutations in the conserved residues of the Walker ‘A’ motif 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. Subcellular fractionation studies of VirB11 truncation derivatives indicated that both the N- and the C-terminal halves of VirB11 contain membrane interaction determinants. Merodiploid analysis demonstrating that a mutant allele with substitution of two amino acids and an allele with a deletion of the first half of the gene had transdominant effects over wild-type virB11, provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 assembles as a homo-multimer in the presence and in the absence of other components of the T-complex transport system. First, native VirB11 and a fusion protein composed of VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage l cI repressor conferred immunity to l superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker ‘A’ motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His6 tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex.
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Studies of Subcellular Localization and Complex Formation of the Agrobacterium Tumefaciens Transport ATPase VirB11