EmrE, a small Escherichia coli multidrug transporter, protects Saccharomyces cerevisiae from toxins by sequestration in the vacuole

Abstract

In this report we describe the functional expression of EmrE, a 110-amino-acid multidrug transporter from Escherichia coli, in the yeast Saccharomyces cerevisiae. To allow for phenotypic complementation, a mutant strain sensitive to a series of cationic lipophilic drugs was first identified. A hemagglutinin epitope-tagged version of EmrE (HA-EmrE) conferring resistance to a wide variety of drugs, including acriflavine, ethidium, methyl viologen, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), was functionally expressed in this strain. HA-EmrE is expressed in yeast at relatively high levels (0.5 mg/liter), is soluble in a mixture of organic solvents, and can be functionally reconstituted in proteoliposomes. In bacterial cells, EmrE removes toxic compounds by active transport through the plasma membrane, lowering their cytosolic concentration. However, yeast cells expressing HA-EmrE take up 14C-methyl viologen as well as control cells do. Thus, we investigated the basis of the enhanced resistance to the above compounds. Using Cu2+ ions or methylamine, we could selectively permeabilize the plasma membrane or deplete the proton electrochemical gradients across the vacuolar membrane, respectively. Incubation of yeast cells with copper ions caused an increase in 14C-methyl viologen uptake. In contrast, treatment with methylamine markedly diminished the extent of uptake. Conversely, the effect of Cu2+ and methylamine on a plasma membrane uptake system, proline, was essentially the opposite: while inhibited by the addition of Cu2+, it remained unaffected when cells were treated with methylamine. To examine the intracellular distribution of HA-EmrE, a functional chimera between HA-EmrE and the green fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE-GFP fluorescence distribution was virtually identical to that of the vacuolar marker FM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplasmic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organisms in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.

FIG. 1

HA-EmrE protects bacteria and yeast cells from a wide range of cytotoxic compounds. (A) Diagrammatic presentation of the generation of HA-EmrE. EmrE was inserted in frame at the BamHI site of the BFG-1 vector, creating the epitope-tagged HA-EmrE. HA-EmrE contains 24 additional amino acids at the N-terminus comprising two repeats of the HA epitope (YPYDVPDYA). (B) HA-EmrE, like the wild-type EmrE, confers resistance to bacterial cells. Five-microliter aliquots of logarithmic dilutions (1:10² to 1:10⁷) from an overnight culture were spotted on LB plates in the absence (control) or presence of either 0.2 mM acriflavine, 0.2 mM methyl viologen, or 0.5 mM ethidium. (C) YAE65 is an S. cerevisiae strain particularly sensitive to MPP⁺. Overnight cultures were tested for susceptibility to the toxin MPP⁺. In comparison to the yeast wild-type strain BWT-1, YHE4 shows an increased sensitivity surpassed only by that of the extremely sensitive isogenic strain YAE65. (D) Yeast cells expressing HA-EmrE show an increased resistance to pleiotropic drugs. YAE65 cells were grown overnight in minimal medium. Serial dilutions (1:1 to 1:10⁵) were performed in sterile water, and 5-μl suspensions were spotted on YPD plates containing the indicated drug concentrations. After 3 days of incubation at 30°C, the plates were photographed.

FIG. 2

HA-EmrE is extractable in a chloroform-methanol mixture and can be functionally reconstituted in proteoliposomes. (A) HA-EmrE is soluble in a chloroform-methanol mixture. Membranes and C:M extracts were produced from yeast cells, separated on a 16% Tricine gel (33), and assayed for protein expression with a monoclonal antibody against the HA epitope. Membrane samples (lanes 1 to 4) and C:M (Chl:Meth) extracts (lanes 5 to 7) consisted of cells transformed with BFG-1 mock vector (control; lane 1), HA-EmrE (lanes 2 and 5), HA-EmrE II (lanes 3 and 6), HA-EmrE-GFP (lane 4 and 7). (B) HA-EmrE can be reconstituted in proteoliposomes. Transport of ¹⁴C-methyl viologen was assayed in proteoliposomes prepared with C:M extracts from either BFG-1- or HA-EmrE-transformed cells as described in Materials and Methods. (C) Substrates of EmrE inhibit the uptake of ¹⁴C-methyl viologen. Uptake of ¹⁴C-methyl viologen was measured in the presence of increasing concentrations of ethidium bromide. The inhibition curve is in agreement with previous studies (45). Similar results were achieved with acriflavine (not shown).

FIG. 3

HA-EmrE catalyzes the compartmentalization of toxins in an acidic intracellular compartment. (A) HA-EmrE-expressing and control (BFG-1) cells take up similar amounts of ¹⁴C-methyl viologen. HA-EmrE-expressing and BFG-1 cells were assayed for total uptake of ¹⁴C-methyl viologen by a rapid filtration method. (B) Permeabilized HA-EmrE-expressing cells take up increased amounts of ¹⁴C-methyl viologen. Uptake of ¹⁴C-methyl viologen was measured on BFG-1 (closed symbols) and HA-EmrE-expressing (open symbols) cells treated with either 0.5 mM CuSO₄ (•, ○) (27) or 10 mM methylamine (■, □) (14) as described in Materials and Methods. (C and D) The effects of Cu²⁺ and methylamine on a plasma membrane transport system are essentially the opposite. HA-EmrE (open symbols)- and BFG-1 (closed symbols)-transformed cells were assayed for the ability to transport [³H]proline in the presence of 0.5 mM CuSO₄ (•, ○) or 10 mM methylamine (■, □) or in the absence of further additions (▵, ▴).

FIG. 4

HA-EmrE-GFP confers resistance to yeast cells and shows a distinct pattern of vacuolar staining. (A) The HA-EmrE-GFP chimera confers resistance to yeast cells against toxic compounds. YAE65 cells transformed with either BFG-1 (control), HA-EmrE, HA-EmrE II, or HA-EmrE-GFP were grown overnight in minimal medium. Serial dilutions (1:1 to 1:10⁴) were performed, and 5-μl aliquots of the suspensions were spotted on YPD plates either lacking or containing 1.5 mM MPP⁺; 3 days later, plates were photographed. (B and C) HA-EmrE-GFP shows a typical vacuolar distribution. Cells expressing HA-EmrE-GFP (B) and HA-GFP (C) were stained for 60 min with 10 μM FM 4-64 (40), washed, and visualized in a confocal microscope as described in Materials and Methods. HA-GFP shows a consistent cytosolic distribution. HA-EmrE-GFP specifically locates in the tonoplast; FM 4-64 and HA-EmrE-GFP images are clearly overlapping.