Purification and reconstitution of an osmosensor: Transporter ProP of Escherichia coli senses and responds to osmotic shifts

The ProP protein of Escherichia coli is an osmoregulatory H+-compatibIe sol ute cotransporter. ProP is activated by an osmotic upshift in both whole ce lls and membrane vesicles. We are using biochemical and biophysical techniq ues to explore the osmosensory and catalytic mechanisms of ProP. We now rep ort the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. c oli galP promoter, and ProP-(His)(6) was shown to be functionally equivalen t to wild-type ProP by enzymatic assay of whole cells. ProP-(His)(6), purif ied by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies ra ised against the ProP protein. ProP-(His)(6) was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The recons tituted transporter mediated proline accumulation only if (1) a membrane po tential was generated by valinomycin-mediated K+ efflux and (2) the proteol iposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by Delta pH. Pure ProP acts, in the proteoliposome environ ment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.