Antiparallel dimers of the small multidrug resistance protein EmrE are more stable than parallel dimers

Abstract

The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.

FIGURE 1.

Topology mapping of EmrE variants by cysteine accessibility. A, schematic representations of the oppositely orientated EmrE variants, EmrE(C_in) and EmrE(C_out). The mutations in EmrE(C_in) are R29G, R82S, and S107K, and in EmrE(C_out) T28R, L85R, and R106A. Black dots represent positively charged residues. The white starburst represents the position of the single cysteine Cys¹⁰⁸ used for topology determination by MTSET/Mal-PEG treatment of whole cells. B, cysteine labeling of EmrE(C_in)^T108C and EmrE(C_out)^T108C. Periplasmic cysteines were blocked by treatment of whole cells with membrane-impermeable MTSET, cells were lysed, and remaining free cysteines were reacted with Mal-PEG that causes a size shift (black dots). The disulfide-bonded dimer of EmrE(C_out)^T108C is indicated by a white dot. C, SDS-PAGE gels showing spontaneous in vivo cysteine cross-linking of EmrE(C_out)^T108C (lane 3, white dot) but not of EmrE^T108C, EmrE(C_in)^T108C, or co-expressed EmrE(C_in)/EmrE(C_out)^T108C (lanes 1, 2, and 4). Lanes 5–8 shows the same samples after reduction with DTT. Note that EmrE(C_in) migrates faster than EmrE(C_out) and EmrE(WT) on SDS-PAGE gels.

FIGURE 2.

Analysis of dimer formation by BN-PAGE. A, BN-PAGE of EmrE(WT) (○), EmrE(C_in) (△), EmrE(C_out) (▿), and co-expressed versions. Monomeric (M), dimeric (D), and tetrameric (T) forms and their composition are indicated. Note that EmrE(C_out) migrates faster than EmrE(C_in) and EmrE(WT) on BN-PAGE, opposite to the situation for SDS-PAGE. B, two-dimensional SDS-gel of BN-PAGE separated samples of EmrE(C_in), EmrE(C_out), co-expressed EmrE(C_in)/EmrE(C_out), and co-expressed EmrE(C_out)/EmrE(C_in). C, SDS-PAGE of the same samples as in A and B (co-expressed EmrE(C_in)/EmrE(C_out) and co-expressed EmrE(C_out)/EmrE(C_in)), illustrating that the relative expression levels depend on the cloning site (MCS1, MCS2) in the vector. The protein encoded by the gene in MCS1 tends to be more highly expressed than the gene in MCS2.

FIGURE 3.

BN-PAGE of EmrE(WT)-MycHis, co-expressed EmrE(WT)-Myc-His/EmrE(C_in), and co-expressed EmrE(WT)-Myc-His/EmrE(C_out). The compositions of the different dimers are indicated by the same symbols as described in the legend to Fig. 2. EmrE(C_in) and EmrE(C_out) are included for comparison.

FIGURE 4.

EmrE(C_in) and EmrE(C_out) homodimers reassemble into a mixture of EmrE(C_in)/EmrE(C_out) heterodimers, EmrE(C_in) homodimers, and EmrE(C_out) homodimers after heating to 60 °C and cooling. A, BN-PAGE of cells expressing EmrE(C_in) and cells expressing EmrE(C_out) that were mixed before lysis (lane 3) or mixed after membrane collection but before solubilization in DDM (lane 4). EmrE(C_in) (lane 1), co-expressed EmrE(C_in)/EmrE(C_out) (lane 2), co-expressed EmrE(C_out)/EmrE(C_in) (lane 5), and EmrE(C_out) (lane 6) are included for comparison. B, BN-PAGE of DDM-solubilized EmrE(C_in) mixed with DDM-solubilized EmrE(C_out) and incubated at either 4 °C or 60 °C for different times as indicated (lanes 3–6). EmrE(C_in) (lane 1), co-expressed EmrE(C_in)/EmrE(C_out) (lane 2), and EmrE(C_out) (lane 7) are included for comparison. C, BN-PAGE of DDM-solubilized EmrE(C_in) mixed with DDM-solubilized EmrE(WT)-Myc-His (lanes 1–4) and of EmrE(C_out) mixed with DDM-solubilized EmrE(WT)-Myc-His (lanes 5–8) incubated at either 4 or 60 °C for different times as indicated. The composition of the different dimers are indicated by the same symbols as described in the legend to Fig. 2.

FIGURE 5.

EmrE(C_in)/EmrE(C_out) heterodimers reassemble into a mixture of EmrE(C_in)/EmrE(C_out) heterodimers, EmrE(C_in) homodimers, and EmrE(C_out) homodimers after heating to 60 °C and cooling. A, schematic figure summarizing the proposed rearrangement of homodimers and the formation of heterodimers after mixing and heating DDM-solubilized EmrE(C_in) and EmrE(C_out). The starting point (I) indicates cells expressing EmrE(C_in) (triangles) and cells expressing EmrE(C_out) (inverted triangles). Solubilization (II) preserves the native organization of the dimers. After mixing of micelles (III), the native organization is preserved (IV), unless the micelles are heated and then cooled (V). Heat treatment results in a 1:2:1 mixture of antiparallel EmrE(C_in) homodimers, EmrE(C_in)/EmrE(C_out) heterodimers, and EmrE(C_out) homodimers. B, BN-PAGE of DDM-solubilized co-expressed EmrE(C_in)/EmrE(C_out) (lanes 2–4) and co-expressed EmrE(C_out)/EmrE(C_in) (lanes 5–7) incubated at 60 °C for the indicated times. Co-expressed but unheated EmrE(C_in)/EmrE(C_out) (lane 1) and co-expressed EmrE(C_out)/EmrE(C_in) (lane 8) samples are included for comparison. C, quantitation of dimer disruption and reassembly of DDM-solubilized samples of separately expressed EmrE(C_in) and EmrE(C_out) (left panel; cf. Fig. 4B) and of co-expressed EmrE(C_in)/EmrE(C_out) (middle panel; cf. Fig. 5B) after incubation at 60 °C for the indicated times and cooling. The fractions of the different dimeric forms are shown as a function of incubation time at 60 °C. Quantitation of the three dimer peaks was done by fitting of three Gaussians to the scanned density in this region of the gel using the QtiPlot software, as in the example shown (right panel); au, arbitrary units. Because of the rather large peak overlaps, the width of each peak was set manually to the same constant value for the three peaks before the fitting to ensure a reproducible fit. 10′, 10 min; 20′, 20 min; 30′, 30 min.