Peptide-specific antibody for the melibiose carrier of Escherichia coli localizes the carboxyl terminus to the cytoplasmic face of the membrane.
The synthetic decapeptide NH2-Cys-Val-Gly-Ala-Val-Ser-Asp-Val-Lys-Ala-COOH (designated MBct10), which corresponds to the carboxyl terminus of the melibiose carrier of Escherichia coli, was synthesized and used to raise antibodies in a rabbit. Anti-MBct10 antibodies recognizes the normal melibiose carrier but not a truncated carrier lacking 14 carboxyl-terminal amino acids. Thus the antibodies are specific for the carboxyl terminus of the carrier and not for other domains of the protein. When right-side-out and inside-out membrane vesicles were probed with anti-MBct10 serum, only the inside-out vesicles bound antibody. The carboxyl terminus of the melibiose carrier protein is therefore exposed on the cytoplasmic surface of the membrane. The co-localization of both NH2- and carboxyl termini to the cytoplasmic surface dictates that the protein cross the membrane an even number of times. These data together with hydrophobicity analysis support a topological model for the melibiose carrier with 10 or 12 transmembrane domains.
Botfield
M C
MC
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
Wilson
T H
TH
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D026901:Membrane Transport Proteins / Q000276:immunology*; D011817:Rabbits; D027981:Symporters
D000906:Antibodies; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease