A thermostable K⁺‐stimulated vacuolar‐type pyrophosphatase from the hyperthermophilic bacterium Thermotoga maritima

Abstract

Current evidence suggests the occurrence of two classes of vacuolar-type H + -translocating inorganic pyrophosphatases (V-PPases) : K + -insensitive proteins, identified in eukaryotes, bacteria and archaea, and K + -stimulated V-PPases, identified to date only in eukaryotes. Here, we describe the functional characterization of a thermostable V-PPase from the anaerobic hyperthermophilic bacterium Thermotoga maritima by heterologous expression in Saccharomyces cerevisiae. The activity of this 71-kDa membrane-embedded polypeptide has a near obligate requirement for K + , like the plant V-PPase, and its thermostability depends on the binding of Mg 2+ . Phylogenetic analysis of protein sequences consistently assigned the T. maritima V-PPase to the K + -sensitive class of V-PPases so far only known for eukaryotes. The finding of a K + -stimulated V-PPase also in a member of a primitive eubacterial lineage strongly supports an ancient evolutionary origin of this group of pyrophosphate-energized proton pumps. ß

RETRACTED: Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H + -translocating pyrophosphatases

“…sPPases have been identified in prokaryotes, plastids, and mitochondria of diverse eukaryotes and the cytosol of animal and fungal cells (4). H ϩ -PPases have been reported to occur in different groups of eubacteria and archaea (5)(6)(7)(8) and in internal (vacuo-lysosomal) membranes of higher plants (5,9,10) and diverse photosynthetic and nonphotosynthetic protists (11,12). Two distinct biochemical subclasses of H ϩ -PPases have been characterized to date: K ϩ -stimulated (3,7,11,12) and K ϩ -insensitive (6,9,14) H ϩ -PPases.…”

Identification of Organelles in Bacteria Similar to Acidocalcisomes of Unicellular Eukaryotes

“…Enzymes from the archeon Pyrobaculum aerophilum (36) the plant Arabidopsis thaliana (AVP2) (37) and the bacterium R. rubrum (38) are capable of PP i hydrolysis in the absence of K ϩ , whereas those from the plants A. thaliana (AVP1) (39) and Vigna radiata (40), the bacteria Thermatoga maritima (41) and Carboxydothermus hydrogenoformans (31), and the trypanosomatid T. cruzi (33) require millimolar concentrations of K ϩ for activity (31). On the basis of phylogenetic analyses of a large set of H ϩ -PPase sequences, it was suggested that K ϩ -dependent and K ϩ -independent enzymes form two independently evolving groups (35,41). A comparison of the amino acid conservation patterns in K ϩ -dependent and K ϩ -independent H ϩ -PPases revealed only two sequence positions where a residue conserved in one type is absent from the equivalent position in all the sequences from the other type (31).…”

The H+-pyrophosphatase of Rhodospirillum rubrum Is Predominantly Located in Polyphosphate-rich Acidocalcisomes

“…Bacteria were post-fixed with OsO 4 for 45 min and then for 15 min with potassium ferricyanide, washed, and treated with 2% uranyl acetate for 30 min. Subsequently, the samples were dehydrated by successive incubations of 6 min with increasing concentrations of ethanol (10,25,50,75,95, and 100%) at room temperature. Epoxy embedding was carried out by resuspending the sample once in 1:1 ethanol/acetonitrile, twice in 100% acetonitrile, then 30 min in 1:1 Epoxy/acetonitrile, 1.5 h in 3:1 Epoxy/acetonitrile, and 4 h in 100% Epoxy.…”

“…Fixed bacteria were washed with Dulbecco's PBS and dehydrated by successive incubations of 6 min each with increasing concentrations of ethanol (10,25,50,75,95, 100, and 100%) at Ϫ20°C. The samples were embedded in Unicryl at 4°C by incubation with 1:1 ethanol/Unicryl for 1 h and 100% Unicryl for 1, 16, and 8 h. The embedded samples were polymerized under UV irradiation at Ϫ20°C for 48 h. Thin sections were collected on 300-mesh nickel grids and blocked for 30 min with PBS containing 0.1% Tween 20 and 0.5% cold fish gelatin (PBS-TW-FG).…”