Orientation of the carboxyl terminus of the Na+/proline symport carrier in Escherichia coll

The orientation of the carboxyl terminal region of the Escherichia coli proline carrier in the cytoplasmic membrane was studied. The β-galactosidase moiety of the PutP-LacZ fusion protein [(1987) J. Biol. Chem. 262, 14100-14104] was exposed outside the inside-out vesicles and inside the right-side-out vesicles. A site-directed antibody raised against a synthetic peptide corresponding to the putative carboxyl terminal region of the carrier reacted preferentially with the inside-out vesicles prepared from a wild-type proline carrier overproducing strain and less with the right-side-out vesicles. These results indicate that the carboxyl terminus of the proline carrier is exposed to the cytoplasmic side of the membrane.Proline carrier, orientation; Carboxyl terminus; Fusion protein, PutP-LacZ; Antibody, site-directed; ELISA, competitive