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

摘要

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophos-phate 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 phylo-genetic 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 hydro-lytic 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.