Fig. 1. Model of EmrE–His with the four transmembrane regions predicted by hydropathy plots. The His and Myc tags are indicated by open ovals with one-letter amino acid codes shown in bold italicized text. Dashed ovals indicate residues that were incorporated into the transporter in the process of linking in the epitope tags. The Glu14 residue in the first transmembrane region is highlighted. The Myc tag is used here only as a linker to keep the His residues away from the membrane. Without the linker, the His-tagged transporter displays only residual activity. Inset, SDS–PAGE analysis of different stages of purification. Lane 1, total membranes; lane 2, detergent-solubilized extract after Ni–NTA purification, unbound fraction; lane 3, EmrE after Ni–NTA purification; lane 4, EmrE after size exclusion purification. The arrow indicates the monomeric form of EmrE; the asterisk the dimeric form. The apparent M_r of monomeric EmrE–His is 14 400 Da.