Functional properties of the purified Na(+)-dependent citrate carrier of Klebsiella pneumoniae: evidence for asymmetric orientation of the carrier protein in proteoliposomes.

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified and reconstituted into liposomes to investigate the properties of this transport system without interference from other proteins. Citrate uptake was an electroneutral process, where delta pNa+ and/or delta pH are driving forces. Delta psi was unable to stimulate citrate transport, either alone or in addition to the other driving forces. Sodium ions on the inside of the proteoliposomes stimulated the uptake of citrate, indicating that Na+ ions recycle during the transport of citrate. CitS also performed Na+ counterflow in the absence of citrate. The citrate carrier performed citrate/citrate counterflow but no heterologous antiport of citrate with one of the end products arising from the anaerobic citrate fermentation pathway (acetate, formate, or bicarbonate) in K. pneumoniae. Citrate counterflow kinetics revealed that CitS transports citrate according to a simultaneous type of mechanism. The Km and Ki values revealed two binding sites for citrate: one with low and one with high affinity. This transport mode is in accord with an asymmetric organization of the carrier protein in proteoliposomes.