Membrane topology of the melibiose carrier of Escherichia coli.
The minimum structural information necessary to formulate and assess mechanistic models of integral membrane protein function is that of membrane topology. This paper characterizes the topological structure of the melibiose carrier of Escherichia coli based on constraints provided by genetic fusions to the compartment-specific reporter protein alkaline phosphatase. Twenty-eight unique chimeras exhibiting either low alkaline phosphatase activity (cytoplasmic location of the fusion joint) or high alkaline phosphatase activity (periplasmic location of the fusion joint) were characterized and used in conjunction with Goldman-Engelman-Steitz hydropathy analysis to model topological structure. The melibiose carrier is predicted to have a cytoplasmic amino terminus, two sets of six transmembrane domains separated by an unusually large cytoplasmic loop ("six-loop-six" arrangement), and a 45-residue cytoplasmic carboxyl tail. Remarkably, the identical six-loop-six arrangement is predicted from the hydrophobicity plots of the H(+)-coupled lactose, arabinose, xylose, and citrate cotransporters of E. coli, the glucose transporter from rat brain, the family of glucose transporters isolated from various human tissues and cell lines, and the human, mouse, and hamster multidrug resistance transporters (Henderson, P.J.F. (1990) Res. Microbiol. 141, 316-328; Maloney, P.C. (1990) Res. Microbiol. 141, 374-383). Such a broad degree of conservation (or convergence) suggests a distinct structural and/or mechanistic advantage associated with the six-loop-six motif. The nature of this advantage is as yet unknown.
Botfield
M C
MC
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Naguchi
K
K
Tsuchiya
T
T
Wilson
T H
TH
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002678:Chimera; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D027981:Symporters
D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D000469:Alkaline Phosphatase