Quaternary structure and functional unit of energy coupling factor (ECF)-type transporters

Abstract

ATP-binding cassette (ABC) transporters mediate transport of diverse substrates across membranes. We have determined the quaternary structure and functional unit of the recently discovered ECF-type (energy coupling factor) of ABC transporters, which is widespread among prokaryotes. ECF transporters are protein complexes consisting of a conserved energizing module (two peripheral ATPases and the integral membrane protein EcfT) and a non-conserved integral membrane protein responsible for substrate specificity (S-component). S-components for different substrates are often unrelated in amino acid sequence but may associate with the same energizing module. Here, the energizing module from Lactococcus lactis was shown to form stable complexes with each of the eight predicted S-components found in the organism. The quaternary structures of three of these complexes were determined by light scattering. EcfT, the two ATPases (EcfA and EcfA'), and the S-components were found to be present in a 1:1:1:1 ratio. The complexes were reconstituted in proteoliposomes and shown to mediate ATP-dependent transport. ECF-type transporters are the smallest known ABC transporters.

FIGURE 1.

Purification of EcfAA′T complexes. Coomassie Blue-stained SDS-polyacrylamide gels showing the purified fractions after nickel-Sepharose and size-exclusion chromatography. a, EcfAA′T was expressed in L. lactis (lane 1) or in E. coli (lane 2). b, EcfAA′T was co-produced with seven S-components in E. coli. BioY2 is not shown but behaved in the same way as BioY. The identities of the S-components were confirmed by Western blotting and detection using anti-STREPII tag antibodies (bottom panel). The Western blot was used only for qualitative purposes, and the amounts of protein loaded on the corresponding SDS-polyacrylamide gel were not the same as on the Coomassie Blue-stained gel.

FIGURE 2.

Subunit stoichiometry of the EcfAA′T-S-component complex. a, SEC-MALLS analysis of the EcfAA′T-NiaX complex. The chromatogram of a size-exclusion chromatography run is shown. The black trace is the signal from the differential refractive index detector. The calculated masses of protein (blue), detergent (green), and total (red) of the protein-detergent micelle are shown in the chromatogram. b, schematic representation of an ECF-type importer. The positions of EcfA and EcfA′ relative to the membrane subunits are not known. S indicates S-component; the black circle indicates substrate.

FIGURE 3.

Transport of [³H]niacin (a) and [³H]riboflavin (b) into proteoliposomes containing EcfAA′T-NiaX and EcfAA′T-RibU, respectively. Error bars indicate the S.E. of three measurements. The proteoliposomes were loaded with 50 mm potassium phosphate, supplemented with 10 mm MgSO₄ and 10 mm ATP (closed circles), 10 mm MgSO₄ only (open circles), or 10 mm MgSO₄ and 10 mm ADP (closed triangles). The pH was 7. The accumulation levels of niacin (at the 3-min time point) and riboflavin (at the 6-min time point) are 8.7- and 26-fold, respectively. The non-zero levels of radioactivity (riboflavin or niacin) counted using the proteoliposomes loaded with Mg-ADP or MgSO₄ only are due to nonspecific binding of the label to the proteoliposomes. Similar levels were observed when proteoliposomes of an unrelated protein were used (the secondary active aspartate transporter Glt_Ph, data not shown).