Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4

Abstract

Summary Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants. VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore. To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes. In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence microscopy, cells probed with anti‐VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location of VirD4 was not dependent on T‐DNA processing, the formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large cytoplasmic region contains a nucleotide‐binding domain. To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer. A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer. Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide‐binding domains are required for polar localization of VirD4. The periplasmic domain mutant VirD4Δ36–61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…Pili are produced both on the lateral sides and on the poles of a bacterium, although in case of the A. tumefaciens T-pili they are more abundantly found at cell poles . Likewise, the Mpf transport apparatus of A. tumefaciens (VirB/VirD4) localizes to the bacterial poles (Atmakuri et al, 2003;Judd et al, 2005;Kumar and Das, 2002). This diVerence of the A. tumefaciens VirB/VirD4 system versus bacterial conjugation systems is also reXected in the observed Fig.…”

“…Addi- Table 2 Interactions between VirB/VirD4 components a B, detected biochemically (in vitro); G, suggested by genetic arrangement; H, detected by two-hybrid interaction analysis; O, suggested by other indirect evidence. Citations: 1 Spudich et al (1996); 15 Anderson et al (1996); 16 Farizo et al (1996); 17 Harris et al (2001); 18 Das et al (1997); 19 Das and Xie (2000); 20 Kumar and Das (2001); 21 Gilmour et al (2003); 22 Llosa et al (2003); 23 Krause et al (2000a); 24 Yeo et al (2000); 25 Hormaeche et al (2002); 26 Schröder et al (2002); 27 Kumar and Das (2002).…”

“…Binding and release of nucleotides, which is possibly triggered by Mg 2+ , may have a mechanistic function related to substrate translocation (Schröder and Lanka, 2003). Mutational analysis of the putative nucleotide binding motifs (Walker A and B 'P-loop' motifs) indicates an essential function of this binding activity for the secretion process (Balzer et al, 1994;Kumar and Das, 2002;Moncalián et al, 1999).…”

“…In A. tumefaciens, the CP (VirD4) localizes to the cellular poles. Polar localization occurs independently of the presence of other Mpf components and requires both the N-terminal periplasmic domain as well as the nucleotide-binding motif of the protein (Kumar and Das, 2002).…”

Biochemical Characterization of Protein Complexes from the Helicobacter pylori Protein Interaction Map

“…Two proteins are extracellular and associated with or are part of a pilus system: VirB2 is the pilus subunit itself, while VirB5 is a minor component of the pilus (23,24). Finally, one ATPase and two ATP-binding proteins appear to power the entire machinery: VirB11, VirB4, and VirD4, respectively (25)(26)(27)(28). In H. pylori, homologs for most of these proteins are encoded by the Cag pathogenicity island and are termed HPXXXX (where XXXX denotes the ORF number as defined in Ref.…”