Topological mapping establishes signal peptide-independent cytoplasmic membrane insertion of bacterial MATE family efflux protein EmmdR.

Enterobacter cloacae ATCC13047 encodes the EmmdR efflux protein, which belongs to the Multidrug And Toxic compound Extrusion (MATE) transporter family (Transporter Classification ID 2.A.66.1). The protein has a predicted existence in the Uniprot database owing to the lack of structure elucidation and experimental evidence of membrane localization. This study experimentally validated the predicted topology of EmmdR and ascertained the role of charged amino acids in membrane insertion. The experimental strategy included qualitative and quantitative assays of dual reporter PhoA-LacZ enzymes coupled with microscopic analysis to ascertain the localization of EmmdR. The topology of periplasmic loop 1 was confirmed by immunofluorescence analysis. The results of this study corroborated the predicted 12 transmembrane segment architecture of EmmdR. Colocalization of green fluorescent protein (GFP) with membrane staining dye FM4-64 confirmed cytoplasmic membrane localization and cytoplasmic orientation of C and N-terminus of EmmdR. Immunofluorescence microscopy of spheroplasts confirmed the periplasmic localization of loop 1. The membrane insertion of EmmdR concurred with the 'positive inside' rule, and deletion of critical positive amino acids in the cytoplasmic loop resulted in altered topology. Thus, this study is the first to report the experimental topology determination and membrane localization of EmmdR, which validates the predicted existence and supports the predicted structure of EmmdR.