title: Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations.
abstract: The eleven predicted gene products of the Agrobacterium tumefaciens virB operon are believed to form a transmembrane pore complex through which T-DNA export occurs. The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fraction of A. tumefaciens. Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J. E. Ward, E. M. Dale, E. W. Nester, and A. N. Binns, J. Bacteriol. 172:5200-5210, 1990). Deletion of the sequences encoding amino acids 22 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA transfer to the plant cell. Western blot (immunoblot) analysis indicated that the observed loss of virulence could not be attributed to a decrease in the steady-state levels of the mutant VirB10 protein. Although the deletion of the single transmembrane domain would be expected to perturb membrane association, VirB10 delta 22-55 was found exclusively in the membrane fraction. Urea extraction studies suggested that this membrane localization might be the result of a peripheral membrane association; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugation. Both wild-type VirB10 and wild-type VirB9 were only partially removed from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble. VirB11 was stripped from the membranes by 6 M urea but not by a more mild salt extraction. The fractionation patterns of VirB9, VirB10, and VirB11 were not dependent on each other or on VirB8 or VirD4. The observed tight association of VirB9, VirB10, and VirB11 with the membrane fraction support the notion that these proteins may exist as components of multiprotein pore complexes, perhaps spanning both the inner and outer membranes of Agrobacterium cells.
authors.affiliation: Department of Biology, Haverford College, Pennsylvania 19041, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Finberg
authors.forename: T R
authors.initials: TR
authors.lastname: Muth
authors.forename: S P
authors.initials: SP
authors.lastname: Young
authors.forename: J B
authors.initials: JB
authors.lastname: Maken
authors.forename: S M
authors.initials: SM
authors.lastname: Heitritter
authors.forename: A N
authors.initials: AN
authors.lastname: Binns
authors.forename: L M
authors.initials: LM
authors.lastname: Banta
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000472:pathogenicity*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry*; D004269:DNA, Bacterial / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009154:Mutation; D010944:Plants / Q000382:microbiology*; D011485:Protein Binding; D017384:Sequence Deletion; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; C036483:T-DNA; D037521:Virulence Factors