A simple approach for large-scale purification and unidirectional reconstitution of the Na+/proline transporter of Escherichia coli (PutP) is described. The procedure is based on the insertion of a highly polar peptide composed of 17 amino acids including a 6His tag at the C-terminus of the transporter. Purification of the hybrid protein is achieved by Ni+-NTA affinity (purity >95%) and ion exchange chromatography (purity >99%). The purified transporter is reconstituted into preformed, detergent-destabilized liposomes. Detergent is removed slowly by adsorption to polystyrene beads. The highest activities [Vmax = 1.1 x 10(3) nmol min-1 (mg of protein)-1] are measured when Triton X100 is used for liposomes destabilization at a concentration corresponding to the onset of lipid solubilization. Site-directed labeling of PutP and site-specific proteolytic cleavage indicate that the transporter is inserted into proteoliposomes in an inside-out orientation. Reconstituted PutP is able to accumulate proline against a concentration gradient in the presence of an inwardly directed electrochemical Na+ or Li+ gradient, while a pH gradient does not affect transport. The apparent proline affinity of PutP in proteoliposomes is similar to the value determined with intact cells. Interestingly however, the apparent Na+ affinity of reconstituted PutP is reduced by a factor of about 25 compared to cells, suggesting a lower cation affinity on the cytosolic side of PutP relative to the outside.

Hydropathy profile analysis of the amino acid sequence of the Na ؉ /proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70 -75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutPalkaline phosphatase (PhoA) and PutP-␤-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 model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino 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 3 Cys, Thr-5 3 Cys) or into putative TM II (Ser-71 3 Cys, Glu-75 3 Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 3 Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate 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 pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure 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 cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na ؉ /solute cotransporter family.

Ser57 in the Na'/proline permease of Escherichiu coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser.57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus, alanine, cysteine, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value. In addition, substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs L-azetidine-2-carboxylate and 3,4-dehydro-~,~-proline. Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18, respectively, relative to wild-type. In contrast, all of the Ser57 replacements analyzed cause only small changes in V,,,, values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein.

With respect to the functional importance attributed to the N-terminal part of the Na(+)/proline transporter of Escherichia coli (PutP), we report here on the structural arrangement and functional dynamics of transmembrane domains (TMs) II and III and the adjoining loop regions. Information on membrane topography was obtained by analyzing the residual mobility of site-specifically-attached nitroxide spin label and by determination of collision frequencies of the nitroxide with oxygen and a polar metal ion complex using electron paramagnetic resonance (EPR) spectroscopy. The studies suggest that amino acids Phe45, Ser50, Ser54, Trp59, and Met62 are part of TM II while Gly39 and Arg40 are located at a membrane-water interface probably forming the cytoplasmic cap of the TM. Also Ala67 and Glu75 are at a membrane-water interface, suggesting a location close to the periplasmic ends of TMs II and III, respectively. Ser71 between these residues is clearly in a water-exposed loop (periplasmic loop 3). Spin labels attached to positions 80, 86, and 91 show EPR properties typical for a TM location (TM III). Leu97 may be part of a structured loop region while Ala107 is clearly located in a water-exposed loop (cytoplasmic loop 4). Finally, spin labels attached to the positions of Asp33 and Leu37 are clearly on the surface of the transporter and are directed into an apolar environment. These findings strongly support the recently proposed 13-helix model of PutP [Jung, H., Rübenhagen, R., Tebbe, S., Leifker, K., Tholema, N., Quick, M., and Schmid, R. (1998) J. Biol. Chem. 273, 26400-26407] and suggest that TMs II and III of the transporter are formed by amino acids Ser41 to Gly66 and Ser76 to Gly95, respectively. In addition to the topology analysis, it is shown that binding of Na(+) and/or proline to the transporter alters the mobility of the nitroxide group at the positions of Leu37 and Phe45. From these findings, it is concluded that binding of the ligands induces conformational alterations of PutP that involve at least parts of TM II and the preceding cytoplasmic loop.