Characterization of the Vibrio cholerae ferrous iron transport system, feo

Feo is the major ferrous iron transport system in prokaryotes and has only been partially characterized, as its assembly and mechanism of transport have not been determined. The feo operon in V. cholerae encodes three proteins, FeoA, FeoB, and FeoC, which are all required for function of the Feo system. FeoA and FeoC are both small cytoplasmic proteins and their function remains unclear. FeoB, thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. However, in this study, a model of the topology of the C-terminal membrane-spanning region of FeoB, based on in vivo labeling experiments, is proposed. Further, through the use of scanning cysteine accessibility mutagenesis, it is determined that the N- and C- termini of FeoB are located in the cytoplasm of V. cholerae. Moreover, epitope-tagged FeoB and FeoC are used to show that these proteins form higher order complexes when cross-linked in vivo in V. cholerae. Further analysis reveals that FeoB simultaneously associates with both FeoA and FeoC to form a large inner membrane complex, an observation that has not been reported previously. It is found that FeoA is required for complex formation, while FeoC is required for wildtype protein levels of FeoB. It is also determined that certain amino acid residues in the GTPase region of FeoB are required for function of the Feo system and for complex formation.