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

Abstract

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.

Assembly and mechanism of a group II ECF transporter

“…Given that the group II S subunits are monomeric in isolation (6,11,12), these observations immediately suggest that the A-A′-T energizing module from Thermotoga contains two separate binding sites for S subunits. These data are consistent with the presence of a dual S subunit in the related Co 2+ transporter (13) as well as studies of the ECF transporter for biotin from R. capsulatus (3). Therefore, we propose that there are two copies of the S subunit in the functional transporter complex.…”

“…Models with one A, A′, T, and S (1 × 4 subunit model, Fig. S1) have been proposed (1,(5)(6)(7), but more recent data suggest the presence of multiple T and S subunits in the functional transporter (3,4). To investigate the relative subunit stoichiometry of the Thermotoga ECF riboflavin transporter, the four genes were coexpressed with distinct His-and FLAG-tagged copies of the same subunit.…”

“…However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between ECF and ABC transporters. In addition, the subunit stoichiometry of ECF transporters is controversial (3)(4)(5)(6), and it is unclear whether the two transporter families share a common mechanism.…”

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…c | Some prokaryotes encode the biotin-specific S component BioY, which can function independently of an ECF module. [34][35][36] , which indicate the presence of two or more EcfT subunits and S components per complex. Although it is possible that these stoichiometric discrepancies are the result of genuine structural divergence between different ECF transporters, another explanation is that two or more complexes that have 1/1/1/1 stoichiometry cluster together in the membrane, giving rise to apparently different stoichiometries.…”

“…Alternatively, it is also possible that the solitary BioY proteins are nonspecifically assisted by a protein other than an ECF module to enable toppling, or that they form oligomers to facilitate toppling. Oligomerization of R. capsulatus BioY has been observed when it is heterologously expressed in E. coli cells, although this protein and other solitary S components are monomeric when purified 24,26,35,50 .…”