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

Abstract

Acidocalcisomes are acidic, calcium storage compartments with a H؉ pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H ؉ -pyrophosphatase (H ؉ -PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H ؉ -PPase with pyrophosphate (PP i ) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4,6-diamino-2-phenylindole staining and identified by using 31 P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PP i and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H ؉ -PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PP i synthesis in the chromatophore membranes.

Arabidopsis Type I Proton-Pumping Pyrophosphatase Expresses Strongly in Phloem, Where It Is Required for Pyrophosphate Metabolism and Photosynthate Partitioning

“…However, in vivo data obtained with the H + -PPase from the proteobacterium Rhodospirillum rubrum were consistent with the capacity of this enzyme to play two distinct roles depending on location and conditions. It can act as an intracellular H + pump in the acidocalcisomes (Seufferheld et al, 2004) or a PP i synthase in the chromatophore membranes during illumination (Baltscheffsky et al, 1966). Bacterial H + -PPases share significant homology with their plant counterparts (Seufferheld et al, 2011).…”

Adaptor Protein-3 (AP-3) Complex Mediates the Biogenesis of Acidocalcisomes and Is Essential for Growth and Virulence of Trypanosoma brucei

“…The finding of enzymes and transporters in the surrounding membrane of these organelles was fundamental in understanding their potential function and origin, and these studies started after their description in trypanosomatid and Apicomplexan parasites (3,9,10). The discovery of acidocalcisome-like organelles in bacteria (11,12) and human platelets (13) suggested that the acidocalcisome is an organelle that either evolved before bacterial and eukaryotic lineages diverged (3) or appeared independently by convergent evolution. Recent work has shown that acidocalcisomes have similarities to lysosome-related organelles (LROs).…”

Kinetics of pyrophosphate-driven proton uptake by acidocalcisomes of Leptomonas wallacei

“…The V-H + -PPase is an electrogenic proton pump that has so far been described in bacteria [26], microorganisms [8,40], plants (reviewed in [41]), and recently found in insects [42], being absent in mammalians. The H + -translocating pyrophosphatase activity characterized here was shown to be located in acidocalcisomes in accordance with previous results found in different trypanosomatids [39].…”

“…Acidocalcisomes are calcium-rich acidic organelles first described in Trypanosoma brucei [6] and Trypanosoma cruzi [12], and then in several other trypanosomatids such as Leishmania [10,13,14], Phytomonas [11,15], Herpetomonas [16], Crithidia, Blastocrithidia, and Leptomonas [17], in apicomplexan parasites such as Plasmodium [18,19] and Toxoplasma [20][21][22], in other unicellular organisms such as Chlamydomonas reinhardtii [23], Dictyostelium discoideum [24], and in the bacteria Agrobacterium tumefaciens [25] and Rhodospirillum rubrum [26]. Acidification of acidocalcisomes is promoted by a V-type proton ATPase (V-H + -ATPase) [27,28] and a V-type pyrophosphatase (V-H + -PPase) [8,10].…”