TOPOLOGY OF THE NA+ PROLINE TRANSPORTER OF ESCHERICHIA-COLI/

Hydropathy profile analysis of the amino acid sequence of the Na+/prol ine transporter of Escherichia coli (PutP) suggests that the protein c onsists of 12 transmembrane domains (TMs) which are connected by hydro philic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol Gen., G enet. 208, 70-75). We have tested this prediction by applying a gene f usion approach in combination with a Cys accessibility analysis and si te-specific proteolysis, Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix mode l. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to t he N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all fi ve native Cys residues (Cys-free PutP) is generated. Subsequently, ami no acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are h ighly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) re acts only with the membrane permeant but not with the impermeant reage nt in intact cells. These results contradict the 12-helix motif and in dicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility p attern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orien tation, is readily cleaved by the endoproteinase AspN before Asp-33 (p L2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp- 356 (cL9), These results suggest a cytosolic location of Asp-33 and Le u-37, thereby implying the formation of an additional TM formed by ami no acids of pL2. Based on these observations, a new secondary structur e model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytop lasm. The 13-helix structure is discussed as a common topological moti f for all members of the Na+/solute cotransporter family.