Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens.

Abstract

The VirB/D4 type IV secretion system of Agrobacterium tumefaciens translocates virulence factors (VirE2, VirF, and the VirD2-T-DNA complex) to plant cells. The membrane-bound translocation machinery consists of 12 proteins (VirB1-11 and VirD4) required for substrate translocation. Protein-protein interactions in the membranes were analyzed after extraction with the mild detergent dodecyl-beta-d-maltoside followed by separation under native conditions. Incubation of the membranes with increasing concentrations of the detergent differentially extracted virulence proteins. Separation of the solubilized proteins by blue native electrophoresis revealed cofractionation between two classes of protein complexes containing VirB7. The first class, consisting of major T-pilus component VirB2 and associated proteins VirB5 and VirB7, comigrated in the low molecular mass portion of the gel of about 100 kDa. The second class contains putative translocation complex core components VirB8, VirB9, and VirB10 in the high molecular mass portion of the gel larger than 232 kDa, as well as VirB7. Solubilized proteins were characterized further by gel filtration chromatography. This procedure separated T-pilus-associated proteins VirB2, VirB5, and VirB7 in the low molecular mass range from the other components of the translocation machinery and the substrates VirE2 and VirD2. Fractionation of VirB7-containing complexes (VirB7-VirB7 homodimers and VirB7-VirB9 heterodimers) suggested that they may link the T-pilus components to the core of the translocation machinery. Based on previously described VirB protein interactions and biochemical analysis of C58 wild type as well as of virB5 and virB6 deletion mutants, a model of T-pilus assembly in A. tumefaciens is suggested.