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

Abstract

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.

Pyrophosphate-Fueled Na+and H+Transport in Prokaryotes

“…The best-characterized Na ϩ ,H ϩ -PPase is that of Bacteroides vulgatus (12). Both transport activities of this protein are inhibited by aminomethylenediphosphonate, a nonhydrolyzable PP i analogue that interacts specifi- cally with membrane PPases (35).…”

“…Both families of membrane PPase had long been thought to transport only H ϩ . Only recently has it become known that in prokaryotes, the K ϩ -dependent family is dominated by PPases that transport Na ϩ (Na ϩ -PPases) (10, 11) or both Na ϩ and H ϩ (Na ϩ ,H ϩ -PPases) (12) and accordingly require Na ϩ for PP i hydrolysis. The determination of the three-dimensional (3D) structures of a plant H ϩ -PPase (14) and a bacterial Na ϩ -PPase (15) has been another recent breakthrough in studies of membrane PPases.…”

“…Among eukaryotes, all plants and many protists contain genes encoding H ϩ -translocating PPase (H ϩ -PPase), whereas animals and fungi do not. Two homologous families of membrane PPases are known, K ϩ independent and K ϩ dependent (10)(11)(12)(13); members of the latter family are activated severalfold in the presence of millimolar concentrations of K ϩ . Both families of membrane PPase had long been thought to transport only H ϩ .…”

Cell Stress by Phosphate of Two Protozoa <i>Tetrahymena thermophile</i> and <i>Tetrahymena pyriformis</i>

“…They produce a proton (H + -PPases) or sodium (Na + -PPases) gradient by coupling the hydrolysis energy of pyrophosphate [28]. However, inorganic pyrophosphatases are rarely studied in protozoa, particularly in ciliates, which are the first used organisms to understand the genetic phenomenon of higher organisms.…”

Structural basis for the reversibility of proton pyrophosphatase

“…[23][24][25] The crystal structures of the Thermotiga maritima Na C -PPase and Vigna radiata H C -PPase have been solved in 3 catalytic states -substrate-bound state (VrH C -PPase:Imidodiphosphate:Mg5 complex) (Fig. 1), resting state (TmNa C -PPase:Ca:Mg), and product-bound state (TmNa C -PPase:Pi 2 :Mg4), but the protein conformation when the ion exit channel is open is yet unavailable.…”