Abstract
Membrane pyrophosphatases (PPases), divided into K(+)-dependent and K(+)-independent subfamilies, were believed to pump H(+) across cell membranes until a recent demonstration that some K(+)-dependent PPases function as Na(+) pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na(+) transport, and H(+) transport activities as well as their K(+) and Na(+) requirements in inner membrane vesicles. Four of these enzymes (from Anaerostipes caccae, Chlorobium limicola, Clostridium tetani, and Desulfuromonas acetoxidans) were identified as K(+)-dependent Na(+) transporters. Phylogenetic analysis led to the identification of a monophyletic clade comprising characterized and predicted Na(+)-transporting PPases (Na(+)-PPases) within the K(+)-dependent subfamily. H(+)-transporting PPases (H(+)-PPases) are more heterogeneous and form at least three independent clades in both subfamilies. These results suggest that rather than being a curious rarity, Na(+)-PPases predominantly constitute the K(+)-dependent subfamily. Furthermore, Na(+)-PPases possibly preceded H(+)-PPases in evolution, and transition from Na(+) to H(+) transport may have occurred in several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na(+)-PPases but shifted to within the membrane or helix 5 in H(+)-PPases. These results contribute to the prediction of the transport specificity and K(+) dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K(+) dependence signature, and position of the membrane-located Glu residue.
Highlights
Hydrolysis of PPi to the active transport of cations across membranes and display no sequence homology to any known protein family
The key determinant of Kϩ dependence is a single amino acid position located near the cytoplasm-membrane interface, which is occupied by Ala in the Kϩ-dependent and Lys in the Kϩ-independent enzymes (2)
inner membrane vesicles (IMV) leakage was not significantly affected by membrane PPase substitutions because, in all tested cases, the IMV supported comparable levels of the ATP-dependent Hϩ gradient generated by E. coli housekeeping enzymes
Summary
Sequence and Phylogenetic Analyses—Membrane PPase protein sequences were retrieved from the NCBI Protein Database with BLAST (21) using Rhodospirillum rubrum Hϩ-PPase (YP_426905) as a query sequence. To assay membrane PPase hydrolytic activity, a thermostated reaction vessel was filled with 25 ml of reaction buffer typically containing 0.1 M MOPS-TMA hydroxide (pH 7.2), 5 mM MgCl2, 158 M TMA4PPi, 40 M EGTA, and variable concentrations of NaCl and KCl. In measurements of Kϩ or Naϩ dependence of PPase activity at a constant concentration (50 mM) of the other ion, TMA hydroxide was replaced with NaOH and KOH, respectively. IMV (0.2–1.2 mg) were incubated for 5 min in 2 ml of buffer (20 mM MOPS-TMA hydroxide (pH 7.2), 5 mM MgCl2, 8 M EGTA, and 2 M 9-amino-6-chloro-2-methoxyacridine) supplemented with 1.5–25. MM NaCl and 0 –100 mM KCl before the reaction was initiated by the addition of 0.3 mM TMA4PPi. The reversibility of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching was tested by disrupting the Hϩ gradient formed in IMV via the addition of 10 mM NH4Cl. PPi-energized Naϩ transport into IMV was assayed by determining the accumulation of 22Naϩ within IMV at 22 °C. The filter was transferred to a microcentrifuge tube, 1 ml of Ultima Gold mixture (PerkinElmer Life Sciences) was added, and the amount of 22Naϩ trapped in IMV was determined via liquid scintillation counting
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
