Skip to main content

Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences


H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

Corresponding author
*Corresponding author: Laboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, c/ Constantino Candeira s/n, 15782 Santiago de Compostela (A Coruña), Spain. Tel: 34981563100. Fax: 34881816070. E-mail:
Hide All
Allen, R. D. and Fok, A. K. (1983). Nonlysosomal vesicles (acidosomes) are involved in phagosome acidification in Paramecium . The Journal of Cell Biology 97, 566570.
Baltscheffsky, H., von Stedingk, L.-V., Heldt, H. W. and Klingenberg, M. (1966). Inorganic pyrophosphate formation in bacterial photophosphorylation. Science 153, 11201122.
Baltscheffsky, M., Schultz, A. and Baltscheffsky, H. (1999). H+-PPases a tightly membrane-bound family. FEBS Letters 457, 527533.
Belogurov, G. A. and Lahti, R. (2002). A lysine substitute for K+ . Journal of Biological Chemistry 277, 4965149654.
Bordier, C. (1981). Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 256, 16041607.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 284–254.
Budiño, B., Lamas, J., Pata, M. P., Arranz, J. A., Sanmartín, M. L. and Leiro, J. (2011). Intraspecific variability in several isolates of Philasterides dicentrarchi (syn. Miamiensis avidus), a scuticociliate parasite of farmed turbot. Veterinary Parasitology 175, 260272.
Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J. and Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55, 611622.
Carystinos, G. D., McDonald, H. R., Monroy, A. F., Dhindsa, R. S. and Poole, R. J. (1995). Vacuolar H+-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiology 108, 641649.
Dauly, C., Perlman, D. H., Costello, C. E. and McComb, M. E. (2006). Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses. Journal of Proteome Research 5, 16881700.
Docampo, R., de Souza, W., Miranda, K., Rohloff, P. and Moreno, S. N. (2005). Acidocalcisomes – conserved from bacteria to man. Nature Reviews Microbiology 3, 251261.
Docampo, R., Ulrich, P., Moreno, S. N. J. (2010). Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes. Philosophical Transactions of Royal Society B 365, 775784.
Drozdowicz, Y. M., Kissinger, J. C. and Rea, P. A. (2000). AVP2, a sequence-divergent, K+ insensitive H+-translocating inorganic pyrophosphatase from Arabidopsis . Plant Physiology 123, 353362.
Drozdowicz, Y. M. and Rea, P. A. (2001). Vacuolar H(+) pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends in Plant Science 6, 206211.
Drozdowicz, Y. M., Shaw, M., Nishi, M., Striepen, B., Liwinski, H. A., Roos, D. S. and Rea, P. A. (2003). Isolation and characterization of TgVP1, a type I vacuolar H+-translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor. The Journal of Biological Chemistry 278, 10751085.
Felsenstein, J. (1985). Confidence limits on phylogenies: and approach using the bootstrap. Evolution 39, 783791.
Fukuda, A., Chiba, K., Maeda, M., Nakamura, A., Maeshima, M., Tanaka, Y. (2004). Effect of salt and osmotic stresses on the expression of gene for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley. Journal of Experimental Botany 55, 585594.
Gaxiola, R. A., Li, J., Undurraga, S., Dang, L. M., Allen, G., Alper, S. L. and Fink, G. R. (2001). Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proceedings of the National Academy of Sciences of the United States of America 98, 1144411449.
Gaxiola, R. A., Palmgren, M. G. and Schumacher, K. (2007). Plant proton pumps. FEBS Letters 581, 22042214.
Harper, J. M., Huynh, M. H., Coppens, I., Parussini, F., Moreno, S. and Carruthers, V. B. (2006). A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex. Molecular Biology of the Cell 17, 45514563.
Hu, X. (2014). Ciliates in extreme environments. Journal of Eukaryotic Microbiology 61, 410418.
Iglesias, R., Paramá, A., Álvarez, M. F., Leiro, J., Fernández, J. and Sanmartín, M. L. (2001). Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain). Diseases of Aquatic Organisms 46, 4755.
Iglesias, R., Paramá, A., Álvarez, M. F., Leiro, J., Aja, C. and Sanmartín, M. L. (2003). In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida). Veterinary Parasitology 111, 1930.
Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. Journal of Bacteriology 153, 163168.
Karlsson, J. (1975). Membrane-bound potassium and magnesium ion-stimulated inorganic pyrophosphatase from roots and cotyledons of sugar beet (Beta vulgaris L.). Biochimica et Biophysica Acta 399, 356363.
Kim, Y., Kim, E. J. and Rea, P. A. (1994). Isolation and characterization of cDNAs encoding the vacuolar H+-pyrophosphatase of Beta vulgaris . Plant Physiology 106, 375382.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111120.
Lerchl, J., Kónig, S., Zrenner, R. and Sonnewald, U. (1995). Molecular cloning, characterization and expression of isoforms encoding tonoplast-bound proton-translocating inorganic pyrophosphatase in tobacco. Plant Molecular Biology 28, 833840.
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T Method. Methods 25, 402408.
Long, A. R., Williams, L. E., Nelson, S. J. and Hall, J. L. (1995). Localization of membrane pyrophosphatase activity in Ricinus communis seedlings. Journal of Plant Physiology 146, 629638.
López-López, O., Fuciños, P., Pastrana, L., Rúa, M. L., Cerdán, M. E. and González-Siso, M. I. (2010). Heterologous expression of an esterase from Thermus thermophilus HB27 in Saccharomyces cerevisiae . Journal of Biotechnology 145, 226232.
Luo, S., Marchesini, N., Moreno, S. N. and Docampo, R. (1999). A plant-like vacuolar H(+)-pyrophosphatase in Plasmodium falciparum . FEBS Letters 460, 217220.
Maeshima, M. (2000). Vacuolar H+-pyrophosphatase. Biochimica et Biophysica Acta 1465, 3751.
Mallo, N., Lamas, J. and Leiro, J. M. (2013). Evidence of an alternative oxidase pathway for mitochondrial respiration in the scuticociliate Philasterides dicentrarchi . Protist 164, 824836.
Mallo, N., Lamas, J., Piazzon, C. and Leiro, J. M. (2015). Presence of a plant-like proton-translocating pyrophosphatase in a scuticociliate parasite and its role as a possible drug target. Parasitology 142, 449462.
Marchesini, N., Luo, S., Rodrigues, C. O., Moreno, S. N. J. and Docampo, R. (2000). Acidocalcisomes and vacuolar H+-pyrophosphatase in malaria parasites. Biochemical Journal 347, 243253.
MacIntosh, M. T., Drozdowicz, Y. M., Laroiya, K., Rea, P. A. and Vaidya, A. B. (2001). Two classes of plant-like vacuolar-type H(+)-pyrophosphatases in malaria parasites. Molecular and Biochemical Parasitology 114, 183195.
Marquez, Y., Höpfler, M., Ayatollahi, Z. and Barta, A. (2015). Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity. Genome Research 25, 9951007.
Mitsuda, N., Enami, K., Nakata, M., Takeyasu, K. and Sato, M. H. (2001). Novel type Arabidopsis thaliana H+-PPase is localized to the Golgi apparatus. FEBS Letters 488, 2933.
Moreno, S. N. J. and Docampo, R. (2009). The role of acidocalcisomes in parasitic protists. Journal of Eukaryotic Microbiology 56, 208213.
Moriyama, Y., Hayashi, M., Yatsushiro, S. and Yamamoto, A. (2003). Vacuolar proton pumps in malaria parasite cells. Journal of Bioenergetics and Biomembranes 35, 367375.
Motamayor, J. C., Mockaitis, K., Schmutz, J., Haiminen, N., Livingstone, D., Cornejo, O., Findley, S. D., Zheng, P., Utro, F., Royaert, S., Saski, C., Jenkins, J., Podicheti, R., Zhao, M., Scheffler, B. E., Stack, J. C., Feltus, F. A., Mustiga, G. M., Amores, F., Phillips, W., Marelli, J. P., May, G. D., Shapiro, H., Ma, J., Bustamante, C. D., Schnell, R. J., Main, D., Gilbert, D., Parida, L. and Kuhn, D. N. (2013). The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color. Genome Biology 14, r53.
Paramá, A., Iglesias, R., Álvarez, M. F., Leiro, J., Aja, C. and Sanmartín, M. L. (2003). Philasterides dicentrarchi (Ciliophora, Scuticociliatida): experimental infection and possible routes of entry in farmed turbot (Scophthalmus maximus). Aquaculture 217, 7380.
Paramá, A., Castro, R., Lamas, J., Sanmartín, M. L., Santamarina, M. T. and Leiro, J. M. (2007). Scuticociliate proteinases may modulate turbot immune responses by inducing apoptosis in pronephric leucocytes. International Journal For Parasitology 37, 8795.
Pérez-Castiñeira, J. R., Gómez-García, R., López-Marqués, R. L., Losada, M. and Serrano, A. (2001). Enzymatic systems of inorganic pyrophosphatase bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornestones? International Microbiology 4, 135142.
Pérez-Castiñeira, J. R., Alvar, J., Ruiz-Pérez, L. M. and Serrano, A. (2002). Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa. Biochemical and Biophysical Research Communications 294, 567573.
Philimonenko, V. V., Janácek, J. and Hozák, P. (2002). LR White is preferable to Unicryl for immunogold detection of fixation sensitive nuclear antigens. European Journal of Histochemistry 46, 359364.
Piazzón, C., Lamas, J., Castro, R., Budiño, B., Cabaleiro, S., Sanmartín, M. L. and Leiro, J. (2008). Antigenic and cross-protection studies on two turbot scuticociliate isolates. Fish and Shellfish Immunology 25, 417424.
Piazzon, C., Lamas, J. and Leiro, J. M. (2011). Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.). Parasite Immunology 33, 535544.
Plattner, H. (2010). Membrane trafficking in protozoa SNARE proteins, H+-ATPase, actin, and other key players in ciliates. International Review of Cell and Molecular Biology 280, 79184.
Plattner, H., Sehring, I. M., Mohamed, I. K., Miranda, K., De Souza, W., Billington, R., Genazzani, A. and Ladenburger, E. M. (2012). Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs parasitic Apicomplexa (Plasmodium, Toxoplasma). Cell Calcium 51, 351382.
Poulos, M. G., Batra, R., Charizanis, K. and Swanson, M. S. (2011). Developments in RNA splicing and disease. Cold Spring Harbor Perspectives in Biology 3, a000778.
Rea, P. A. and Poole, R. J. (1993). Vacuolar H+-translocanting pyrophosphatase. Annual Review of Plant Physiology and Plant Molecular Biology 44, 157180.
Rea, P. A., Kim, Y., Sarafian, V., Poole, R. J., Davies, J. M. and Sanders, D. (1992). Vacuolar H+-translocating pyrophosphatases: a new category of ion translocase. Trends in Biochemical Sciences 17, 348353.
Robinson, D. G., Haschke, H. P., Hinz, G., Hoh, B., Maeshima, M. and Marty, F. (1996). Immunological detection of tonoplast polypeptides in the plasma membrane of pea cotyledons. Planta 198, 95103.
Saitoh, O., Murata, Y., Odagiri, M., Itoh, M., Itoh, H., Misaka, T. and Kubo, Y. (2002). Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling. Proceedings of the National Academy of Sciences of the United States of America 99, 1013810143.
Saliba, K. J., Allen, R. J., Zissis, S., Bray, P. G., Ward, S. A. and Kirk, K. (2003). Acidification of the malaria parasite's digestive vacuole by a H+-ATPase and a H+-pyrophosphatase. The Journal of Biological Chemistry 278, 56055612.
Sarafian, V., Kim, Y., Poole, R. J. and Rea, P. A. (1992). Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana . Proceedings of the National Academy of Sciences of the United States of America 89, 17751779.
Scott, D. A. and Docampo, R. (2000). Characterization of isolated acidocalcisomes of Trypanosoma cruzi . The Journal of Biological Chemistry 275, 2421524221.
Serrano, A., Pérez-Castiñeira, J. R., Baltscheffsky, M. and Baltscheffsky, H. (2007). H+-PPases: yesterday, today and tomorrow. IUBMB Life 59, 7683.
Serrano, A., Pérez-Castiñeira, R., and Baltscheffsky, H. (2004). Proton-pumping inorganic pyrophosphatases in some archea and other extremophilic prokaryotes. Journal of Bioenergetics and Biomembranes 36, 127133.
Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., López, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J. D. and Higgins, D. G. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7, 539.
Silva, P. and Gerós, H. (2009). Regulation by salt of vacuolar H+-ATPase and H+-pyrophosphatase activities and Na+/H+ Exchange. Plant Signaling and Behavior 4, 718726.
Scott, D. A., de Souza, W., Benchimol, M., Zhong, I., Lu, H. G., Moreno, S. N. and Docampo, R. (1998). Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi. The Journal of Biological Chemistry 273, 2215122158.
Sakakibara, Y., Kobayashi, H. and Kasamo, K. (1996). Isolation and characterization of cDNAs encoding vacuolar H+-pyrophosphatase isoforms from rice (Oryza sativa L.). Plant Molecular Biology 31, 10291038.
Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 27252729.
Venter, M., Gronewald, J.-H. and Botha, F. C. (2006). Sequence analysis and transcriptional profilling of two vacuolar H+-pyrophosphatase isoforms in Vitis vinífera . Journal of Plant Research 119, 469478.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 0031-1820
  • EISSN: 1469-8161
  • URL: /core/journals/parasitology
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed