A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

Abstract

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H + uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H + -pyrophosphatase inhibitors. H + uptake was not promoted by ATP. Addition of 50 M Ca 2+ induced the release of H + , suggesting the presence of a Ca 2+ /H + countertransport system in the membranes of the acidic compartments. Na + was unable to release protons from the organelles. The pyrophosphate-dependent H + uptake was dependent of ion K + and inhibited by Na + Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H + -pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles.Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H + -pyrophosphatase and a Ca 2+ /H + antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H + transport in the acidocalcisomes of this parasite.

New Insights into Roles of Acidocalcisomes and Contractile Vacuole Complex in Osmoregulation in Protists

“…The isolation method for these organelles was improved (Salto et al, 2008; Scott and Docampo, 2000; Yagisawa et al, 2009); acidocalcisomes were isolated and characterized in other trypanosomatids (Mendoza et al, 2002; Miranda et al, 2004a; Miranda et al, 2004b; Miranda et al, 2004c; Moraes Moreira et al, 2005; Soares Medeiros et al, 2005), Apicomplexan parasites (Marchesini et al, 2000; Moreno and Zhong, 1996; Ruiz et al, 2004b; Soares Medeiros et al, 2011), Chlamydomonas reinhardtii (Ruiz et al, 2001a), D. discoideum (Marchesini et al, 2002), the bacteria Agrobacterium tumefaciens (Seufferheld et al, 2003) and Rhodospirillum rubrum (Seufferheld et al, 2004), human platelets (Ruiz et al, 2004a), mast cells (Moreno-Sanchez et al, 2012), insect (Motta et al, 2009; Ramos et al, 2011), chicken (Ramos et al, 2010b), and sea urchin (Ramos et al, 2010a) eggs; their chemical composition investigated (Ferella et al., 2008; Salto et al, 2008); a number of pumps, channels and exchangers in their membranes were biochemically characterized and their genes cloned, and expressed (Fang et al, 2007; Huang, 2013; Montalvetti et al, 2004; Rohloff et al, 2004); their biogenesis was studied (Besteiro et al, 2008; de Jesus et al., 2010; Huang et al, 2011; Madeira da Silva and Beverley, 2010); and the investigation of their functional roles was started (Docampo et al, 2011). Some of these studies will be the subject of this review.…”

Volutin Granules of Eimeria Parasites are Acidic Compartments and Have Physiological and Structural Characteristics Similar to Acidocalcisomes

“…Antibodies raised against a conserved domain (326 peptide) of A. thaliana vacuolar proton pyrophosphatase (V‐H + ‐PPase) have been shown to recognize acidocalcisomes in a number of parasites (Miranda et al 2004c; Soares Medeiros et al 2005). To confirm that the 326 domain was conserved in Eimeria parasites, sequence alignments of the 326 peptide from A. thaliana V‐H + ‐PPase with Eimeria sequences were carried out.…”

“…The advantage of this type of preparation is the observation of the whole parasite (and whole organelles) without the possible side effects of fixatives and other chemicals used in the routine processing necessary for TEM. This reduces significantly the extraction of the mineral content from the acidocalcisomes and, therefore, allows the qualitative detection of several cations within the organelle by analytical TEM (Miranda et al 2000, 2004a, b, c, 2008; Moreira et al 2005; Soares Medeiros et al 2005). In addition, as cells are imaged as a whole mount, several morphometric parameters such as the number of acidocalcisomes per cell, their shape and mean volume is carried out with higher precision.…”

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

“…Differences in kinetic properties of H þ -PPases from different organisms have been already observed for trypanosomatids. It is possible that those differences might be associated to species-specific characteristics (Rodrigues et al, 1999a, b;Soares Medeiros et al, 2005;Moraes Moreira et al, 2005). The idea that H þ -PPases represent alternative ways to generate proton electrochemical gradients across the membrane of organelles during germination in plant models has been extensively discussed (Maeshima, 2000).…”

“…To investigate whether the activity detected in the previous experiments was preferentially located in the small vesicles, polyclonal antibodies raised against a peptide sequence of A. thaliana H þ -PPase, which was previously shown to cross-react with the H þ -PPase of several trypanosomatids Miranda et al, 2004;Soares Medeiros et al, 2005;Moraes Moreira et al, 2005) and with YGs of the insect R. prolixus (Motta et al, 2004), were used and the material was tested by western blot analysis and immunofluorescence microscopy. Fig.…”