Topologically random insertion of EmrE supports a pathway for evolution of inverted repeats in ion-coupled transporters

Abstract

Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats. This family includes both homodimers and closely related heterodimers. In the former, the topology determinants, evidently identical in each protomer, are weak, and we show that for EmrE, an homodimer from Escherichia coli, the insertion into the membrane is random, and dimers are functional whether they insert into the cytoplasmic membrane with the N- and C-terminal domains facing the inside or the outside of the cell. Also, mutants designed to insert with biased topology are functional regardless of the topology. In the case of EbrAB, a heterodimer homologue supposed to interact antiparallel, we show that one of the subunits, EbrB, can also function as a homodimer, most likely in a parallel mode. In addition, the EmrE homodimer can be forced to an antiparallel topology by fusion of an additional transmembrane segment. The simplicity of the mechanism of coupling ion and substrate transport and the few requirements for substrate recognition provide the robustness necessary to tolerate such a unique and unprecedented ambiguity in the interaction of the subunits and in the dimer topology relative to the membrane. The results suggest that the small multidrug transporters are at an evolutionary junction and provide a model for the evolution of structure of transport proteins.

FIGURE 1.

Tags introduce bias in topology of undecided proteins. Tagged (A) and untagged (B) EmrE K22C and EmrE H110C were metabolically labeled with [³⁵S]methionine in E. coli BW25113 ΔemrEΔmdfA cells and treated with MTSES at the indicated concentrations as described under “Experimental Procedures.” The unreacted thiols were estimated from the degree of reaction with Mal-PEG. EmrE that reacted with Mal-PEG displays a higher apparent M_r in SDS-PAGE, and the ratios of reacted over nonreacted protein were calculated. The bottom panels in A and B are samples of the types of changes at one concentration of MTSES: 0.3 and 0.1 mm, respectively. C, simplified flow chart of procedure. D, monomers of tagged and untagged EmrE K22C (not shown) and H110C are in a parallel topology relative to each other as judged from the full cross-linking detected with o-PDM.

FIGURE 2.

A, schematic representation of the EmrE C_in and EmrE C_out mutations. The mutations were performed as described under “Experimental Procedures.” The sites were selected as in Ref. . The squares indicate positive charges in the wild type EmrE and those remaining after mutagenesis; white circles indicate positive charges added. B, phenotype of EmrE C_in and EmrE C_out. E. coli BW25113 ΔemrE, ΔmdfA, or ΔemrEΔmdfA transformed with pACYC184-EmrE (EmrE), pACYC184 (vector), or pACYC184 with each of the EmrE mutants were grown overnight at 37 °C in LB medium containing chloramphenicol. 5 μl of serial dilutions of the culture were spotted on LB plates containing 30 mm BisTris propane, pH 7.0, with 200 μm IPTG and with or without the addition of ethidium (400 μg/ml in top and middle panels and 100 μg/ml in bottom panel), acriflavine (250, 100, and 25 μg/ml, respectively), and methyl viologen (0.6, 0.4, and 0.3 mm, respectively). Growth was analyzed after overnight incubation at 37 °C. In control plates with no toxicants, growth of all strains was similar (not shown). When resistance was tested with the same strains without IPTG, the phenotypes were negative, and with different host strains the results are variable (shown in Fig. S2), stressing again the inadequacy of a negative phenotype to determine the lack of activity of EmrE or similar proteins.

FIGURE 3.

EbrAB, a heterodimer that can still function as a homodimer. A, schematic representation of EbrAB. The squares represent the positive charges in each monomer. B, phenotype. E. coli BW25113 ΔacrA, ΔmdfA, ΔemrE, Δsmr, or ΔemrEΔmdfA transformed with pACYC184-EmrE (EmrE), pACYC184 (vector), or pACYC184 with EbrA, EbrB, or both genes together were grown overnight at 37 °C in LB medium containing chloramphenicol. 5 μl of serial dilutions of the culture were spotted on LB plates containing 30 mm BisTris propane, pH 7.0, with 200 μm IPTG and with or without the addition of ethidium (ΔacrA, 12.5 μg/ml; ΔmdfA, ΔemrE, and Δsmr, 400 μg/ml; and ΔemrEΔmdfA, 100 μg/ml). Growth was analyzed after overnight incubation at 37 °C. In control plates with no toxicants, growth of all strains was similar (not shown).

FIGURE 4.

A, schematic representation of GpA-EmrE, an EmrE tandem with nine transmembrane helices. GpA-EmrE is shown schematically; transmembrane helices are drawn as boxes. The N terminus of the second EmrE protomer was connected to the C terminus of the first EmrE protomer with a linker derived from a mutant of human GpA (dark box) that does not dimerize (28) and six additional hydrophilic amino acids as described under “Experimental Procedures.” The Myc-His tag is fused to the C terminus of the second monomer. B, growth phenotype of cells expressing GpA-EmrE. E. coli BW25113 ΔemrEΔmdfA cells transformed with either pT7-7-EmrE (EmrE), pT7-7 (vector), or pT7-7-GpA-EmrE (GpA-EmrE) were grown overnight at 37 °C in LB medium with ampicillin. 5 μl of serial dilutions of the culture were spotted on the LB plates containing 30 mm BisTris propane, pH 7.0, with or without 100 μg/ml ethidium, 25 μg/ml acriflavine, or 0.3 mm methyl viologen. Growth was analyzed after overnight incubation at 37 °C.

FIGURE 5.

A, GpA-EmrE does not take part in the intermolecular interactions with other EmrE molecules. Negative dominance of an inactive mutant (EmrE E14C) on EmrE activity and the lack of effect on GpA-EmrE activity were demonstrated as follows. Increasing amounts of membranes containing the indicated amounts of inactive EmrE mutant E14C were solubilized in 1% dodecyl-β-d-maltoside/sodium buffer and mixed with solubilized membranes containing 40 ng of EmrE (●) or GpA-EmrE (▴). After incubation at 80 °C (10 min), the mixture was transferred to 4 °C and assayed for [³H]TPP⁺ binding as described under “Experimental Procedures.” B, probing the packing of GpA-EmrE. Membranes bearing ³⁵S-radiolabeled GpA-EmrE (lanes 1–4) and E22EMH (lanes 5–8) were incubated with the corresponding proteases (T, trypsin; C, chymotrypsin; PK, proteinase K) as described in Ref. . A cartoon is shown indicating the proteolysis sites of the different enzymes: E22EMH is digested by chymotrypsin (after Phe¹¹² in the linker after monomer 1 and the corresponding residue after monomer 2) and trypsin (after Lys¹¹⁹ as above) to produce two polypeptides with a similar molecular mass (11–12-kDa apparent mass in SDS-PAGE) as a result of the digestion of the linker. Proteinase K digests all the linker and the tag. Under the conditions used, the released tag was not detectable; only a minor uncut and tag-less fraction of E22EMH is detectable. In the case of GpA-EmrE only the tag is digested producing a polypeptide of ∼22–24 kDa. The very minor low molecular weight bands observed are present also in the absence of added proteases and may represent small amounts of unrelated proteins.

FIGURE 6.

Topology of GpA-EmrE. BW25113 ΔemrEΔmdfA cells bearing (A) EmrE K22C (♦) and EmrE H110C (•) or (B) GpA-EmrE K22C in the first (■) or the second (▴) monomer were treated with MTSES at the indicated concentrations, and the unreacted thiols were estimated from the degree of reaction with N-ethylmaleimide-fluorescein as described under “Experimental Procedures.” A simplified flow chart is shown in the right panel.