title: Orientation of small multidrug resistance transporter subunits in the membrane: correlation with the positive-inside rule.
abstract: Small multidrug resistance (SMR) transport proteins provide a model for the evolution of larger two-domain transport proteins. The orientation in the membrane of 27 proteins from the SMR family was determined using the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology). Eighteen members were encoded in pairs on the chromosome and shown to insert in fixed orientations; the two proteins in each pair invariably had opposite orientations in the membrane. Interaction between the two proteins in pairs was demonstrated by copurification. The orientation in the membrane of either protein in the pair was affected only marginally by the presence of the other protein. For the proteins in pairs, the orientation in the membrane correlated well with the distribution of positively charges residues (R+K) over the cytoplasmic and extracellular loops (positive-inside rule). In contrast, dual-topology insertion of the singles was predicted less well by the positive-inside rule. Three singles were predicted to insert in a single orientation with the N-terminus and the C-terminus at the extracellular side of the membrane. Analysis of charge distributions suggests the requirement of a threshold number of charges in the cytoplasmic loops for the positive-inside rule to be of predictive value. It is concluded that a combined analysis of gene organization on the chromosome and phylogeny is sufficient to distinguish between fixed or dual topology of SMR members and, probably, similar types of membrane proteins. The positive-inside rule can be used to predict the orientation of members in pairs, but is not suitable as a sole predictor of dual topology.
authors.affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: Magdalena A
authors.initials: MA
authors.lastname: Kolbusz
authors.forename: Ramon
authors.initials: R
authors.lastname: ter Horst
authors.forename: Dirk-Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Juke S
authors.initials: JS
authors.lastname: Lolkema
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D021122:Protein Subunits
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli