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

In this report we describe the functional expression of EmrE, a 110-amino-a cid multidrug transporter from Escherichia coli, in the yeast Saccharomyces cerevisiae. To allow for phenotypic complementation, a mutant strain sensi tive to a series of cationic lipophilic drugs was first identified. A hemag glutinin 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 exp ressed in this strain. HA-EmrE is expressed in yeast at relatively high lev els (0.5 mg/liter), is soluble in a mixture of organic solvents, and can he functionally reconstituted in proteoliposomes. In bacterial cells, EmrE re moves toxic compounds hy active transport through the plasma membrane, lowe ring their cytosolic concentration. However, yeast cells expressing HA-EmrE take up C-14-methyl viologen as well as control cells do. Thus, we investi gated the basis of the enhanced resistance to the above compounds. Using Cu 2+ ions or methylamine, we could selectively permeabilize the plasma membra ne or deplete the proton electrochemical gradients across the vacuolar memb rane, respectively. Incubation of yeast cells,vith copper ions caused an in crease in C-14-methyl viologen uptake. In contrast, treatment with methylam ine markedly diminished the extent of uptake. Conversely, the effect of Cu2 + and methylamine on a plasma membrane uptake system, proline, was essentia lly the opposite: while inhibited by the addition of Cu2+, it remained unaf fected when cells were treated with methylamine. To examine the intracellul ar distribution of HA-EmrE, a functional chimera between HA-EmrE and the gr een fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE- GFP fluorescence distribution was virtually identical to that of the vacuol ar marker PM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplas mic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organism s in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.