THE VIRB4 ATPASE OF AGROBACTERIUM-TUMEFACIENS IS A CYTOPLASMIC MEMBRANE-PROTEIN EXPOSED AT THE PERIPLASMIC SURFACE

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic me mbrane independently of other products of the Ti plasmid. VirB4 was re sistant to extraction from membranes of wild-type strain A348 or a Ti- plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, w hich encodes a periplasmically active alkaline phosphatase (AP) delete d of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP ac tivity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and betwe en residues 450 and 514 (region 2). Conversely, VirB4::beta-galactosid ase hybrid proteins with junction sites mapping to regions 1 and 2 exh ibited low beta-galactosidase activities and hybrid proteins,vith junc tion sites elsewhere exhibited high beta-galactosidase activities. Enz ymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K tr eatment of A. tumefaciens spheroplasts resulted in the disappearance o f the 87-kDa VirB4 protein and the concomitant appearance of two immun oreactive species of similar to 35 and similar to 45 kDa. Taken togeth er, our data support a model in which VirB4 is topologically configure d as an integral cytoplasmic membrane protein with two periplasmic dom ains.