SUBCELLULAR-LOCALIZATION OF 7 VIRB PROTEINS OF AGROBACTERIUM-TUMEFACIENS - IMPLICATIONS FOR THE FORMATION OF A T-DNA TRANSPORT STRUCTURE

Plant cell transformation by Agrobacterium tumefaciens involves the tr ansfer of a single-stranded DNA-protein complex (T-complex) from the b acterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fra ctionation methods were employed to separate inner and outer membranes . Seven VirB-specific antibodies were used on Western blots (immunoblo ts) to detect the proteins in the inner and outer membranes and solubl e (containing cytoplasm and periplasm) fractions. VirB5 was in both th e inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and Vir B10, were present in both inner and outer membrane fractions regardles s of the fractionation method used. Three additional VirB proteins, Vi rB1, VirB4, and VirB11, were found mainly in the inner membrane fracti on by one method and were found in both inner and outer membrane fract ions by a second method. These results confirm the membrane localizati on of seven VirB proteins and strengthen the hypothesis that VirB prot eins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and ou ter membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two memb rane fractions reflect their differential sensitivity to the experimen tal conditions.