Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli.

Abstract

The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside quenches the fluorescence of 2-(4'-maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single-Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM. Unlike full-length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS. the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule.