Subunit composition of an energy-coupling-factor-type biotin transporter analysed in living bacteria

BioMNY a bacterial high-affinity biotin transporter is a member of the recently defined class of ECF (energy-coupling factor) transporters These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry The quaternary structure of BioY, which functions as a low affinity biotin transporter in the absence of BloMN, and of BioMNY was investigated by a FRET (Forster resonance energy transfer) approach using living recombinant Escherichia colt cells To this end, the donor acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities Specific donor acceptor interactions were characterized by lifetime-based FRET spectroscopy The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria