Two independent evolutionary routes to Na+/H+ cotransport function in membrane pyrophosphatases

Abstract

Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi) to transport H+, Na+ or both and help organisms to cope with stress conditions, such as high salinity or limiting nutrients. Recent elucidation of mPPase structure and identification of subfamilies that have fully or partially switched from Na+ to H+ pumping have established mPPases as versatile models for studying the principles governing the mechanism, specificity and evolution of cation transporters. In the present study, we constructed an accurate phylogenetic map of the interface of Na+-transporting PPases (Na+-PPases) and Na+- and H+-transporting PPases (Na+,H+-PPases), which guided our experimental exploration of the variations in PPi hydrolysis and ion transport activities during evolution. Surprisingly, we identified two mPPase lineages that independently acquired physiologically significant Na+ and H+ cotransport function. Na+,H+-PPases of the first lineage transport H+ over an extended [Na+] range, but progressively lose H+ transport efficiency at high [Na+]. In contrast, H+-transport by Na+,H+-PPases of the second lineage is not inhibited by up to 100 mM Na+. With the identification of Na+,H+-PPase subtypes, the mPPases protein superfamily appears as a continuum, ranging from monospecific Na+ transporters to transporters with tunable levels of Na+ and H+ cotransport and further to monospecific H+ transporters. Our results lend credence to the concept that Na+ and H+ are transported by similar mechanisms, allowing the relative efficiencies of Na+ and H+ transport to be modulated by minor changes in protein structure during the course of adaptation to a changing environment.