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 BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli 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.
Finkenwirth
Friedrich
F
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Germany.
Neubauer
Olivia
O
Gunzenhäuser
Julia
J
Schoknecht
Janna
J
Scolari
Silvia
S
Stöckl
Martin
M
Korte
Thomas
T
Herrmann
Andreas
A
Eitinger
Thomas
T
D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D055503:Protein Multimerization; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D021381:Protein Transport; D027981:Symporters / Q000032:analysis* / Q000378:metabolism
D021122:Protein Subunits; D027981:Symporters; C119981:biotin transporter