UNIPORT OF ANIONIC CITRATE AND PROTON CONSUMPTION IN CITRATE METABOLISM GENERATES A PROTON MOTIVE FORCE IN LEUCONOSTOC-OENOS

The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (Delta Psi) and a pH gradient (Delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles oft. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of Delta pH-driven citrate uptake over a range of external pi-Is suggests that the monovalent anionic species (H(2)cit(-)) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H(2)cit(-) is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H+-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).