Membrane-integral pyrophosphatase subfamily capable of translocating both Na ⁺ and H ⁺

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H ⁺ -transporting (found in prokaryotes, protists, and plants) and Na ⁺ -transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and ²² Na ⁺ measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H ⁺ and Na ⁺ into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na ⁺ ,H ⁺ -PPase subfamily, which apparently evolved from Na ⁺ -PPases. Interestingly, genes for Na ⁺ ,H ⁺ -PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na ⁺ ,H ⁺ -PPases require Na ⁺ for hydrolytic and transport activities and are further activated by K ⁺ . Based on ionophore effects, we conclude that the Na ⁺ and H ⁺ transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na ⁺ ,H ⁺ -PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na ⁺ and H ⁺ can be transported via the same mechanism, with switching between Na ⁺ and H ⁺ specificities requiring only subtle changes in the transporter structure.