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

Abstract

In this work, we show the kinetics of pyrophosphate-driven H + uptake by acidocalcisomes in digitonin-permeabilized promastigotes of Leptomonas wallacei. The vacuolar proton pyrophosphatase activity was optimal in the pH range of 7.5-8.0, was inhibited by imidiodiphosphate, and was completely dependent on K + and PPi. H + was released with the addition of Ca 2+ , suggesting the presence of a Ca 2+ /H + antiport. In addition, X-ray elemental mapping associated with energy-filtering transmission electron microscopy showed that most of the Ca, Na, Mg, P, K, Fe, and Zn were located in acidocalcisomes. L. wallacei immunolabeled with antibodies against Trypanosoma cruzi pyrophosphatase show intense fluorescence in cytoplasmatic organelles of size and distribution similar to the acidocalcisomes. Altogether, the results show that L. wallacei acidocalcisomes possess a H + -pyrophosphatase with characteristics of type I V-H + -PPase. However, we did not find any evidence, either for the presence of H + -ATPases or for Na + /H + exchangers in these acidocalcisomes. Ó 2005 Elsevier Inc. All rights reserved. The trypanosomatidae family comprises unicellular eukaryotes that are able to infect a broad range of organisms such as mammalians, fishes, plants, and insects causing a wide spectrum of diseases of social, economical, and medical importance. Leptomonas wallacei is a trypanosomatid parasite that was first isolated by Romeiro et al. [1] from the intestinal tract of the phytophagous hemipteran Oncopeltus fasciatus and since then has been used as a model for studies of the interaction between trypanosomatids and the midgut of the insect vector [2,3]. A remarkable characteristic of these parasites is their ability to form cysts. They occur mainly as promastigotes but can differentiate into cystic forms, which suggests that they represent latent forms of the parasite, protecting it from extreme environmental conditions [1], which include variations on the concentration of ionic species, osmotic pressure, and pH, faced by the parasite in the different portions of the insect gut during the course of infection.Control of intracellular pH in trypanosomatids occurs through the action of proton uptake/release mechanisms located either in the plasma membrane or in intracellular compartments. These include electrogenic proton pumps, proton/ion exchangers, and HCO 3 À and chloride channels [4]. The electrogenic proton pumps comprise the P-type proton ATPase (P-ATPase) 0006-291X/$ -see front matter Ó

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.…”

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.…”

First-Principles Calculations of the Structural, Electronic, Optical, and Mechanical Properties of 21 Pyrophosphate Crystals

“…There are two unrelated families of inorganic pyrophosphatases, the ubiquitous Mg-dependent family, and the Mn-dependent family, and both of them can be found in bacteria and archaea [49,50]. The soluble pyrophosphatases have several functions, including some steroid biosynthetic pathways (such as juvenile hormone synthesis), regulation of cell motility, nucleotide metabolism, and they may have a nuclear role in regulating transcription of some genes [51][52][53][54]. PPases need divalent metal cations to be more active.…”