Acidocalcisomes Are Functionally Linked to the Contractile Vacuole of Dictyostelium discoideum

Abstract

The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H ؉ -ATPase, an H ؉ -pyrophosphatase, and a Ca 2؉ -ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H ؉ -pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dosedependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4-diisothiocyanatodihydrostilbene-2, 2-disulfonic acid and the vacuolar H ؉ -ATPase inhibitor bafilomycin A 1 . Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.

A Functional Aquaporin Co-Localizes with the Vacuolar Proton Pyrophosphatase to Acidocalcisomes and the Contractile Vacuole Complex of Trypanosoma cruzi

“…Immunofluorescence Microscopy-For immunofluorescence, cells were fixed with 4% paraformaldehyde, adhered to poly-l-lysine cover-slips, permeabilized for 5 min with PBS/0.3% Triton X-100, blocked for one hour with PBS/3% bovine serum albumin/5% goat serum/50 mM NH 4 Cl, and incubated with primary antibody for 1 h and then secondary antibody for 45 min. For visualization of the vacuolar H ϩ -pyrophosphatase, mouse polyclonal anti-T. cruzi pyrophosphatase was used at 1:200, followed by goat anti-mouse Alexa 488 or Alexa 546 at 1:1000.…”

“…Acidocalcisomes are also found in a diverse range of trypanosomatid and apicomplexan parasites (2), in the green algae Chlamydomonas reinhardtii (3), in the slime mold Dictyostelium discoideum (4), and in the bacterium Agrobacterium tumefaciens (5). They are characterized, in addition to their acidic nature, by their high density (both in weight and by electron microscopy), and high content of pyrophosphate (PP i ), polyphosphate (polyP), calcium, magnesium, and other elements.…”

“…A role for acidocalcisomes in the response of Leishmania major promastigotes to osmotic stress has also been proposed on the basis of their changes in sodium and chlorine content after hyposmotic stress (7). A functional link between acidocalcisomes and the contractile vacuole complex of C. reinhardtii (3) and D. discoideum (4), which is involved in water extrusion under hyposmotic stress, has also been proposed. In this regard, early observations (8) of epimastigotes of T. cruzi, through phase contrast microscopy, have detected the presence of a contractile vacuole complex as a group of small vacuoles, which fuse as they fill.…”

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

“…These were first described in Trypanosoma brucei [8] and Trypanosoma cruzi [9], then in Leishmania amazonensis [10], Leishmania donovani [11], Plasmodium berghei [12], Plasmodium falciparum [13], Toxoplasma gondii [14] and in organisms such as Chlamydomonas reinhardtii [15], Dictyostelium discoideum [16] and more recently in the bacterium Agrobacterium tumefaciens [17]. These acidic organelles are electron dense, posses a surrounding membrane, have variable size, ranging from 200 ± S.E.…”

<i>PHO8</i> gene coding alkaline phosphatase of <i>Saccharomyces cerevisiae</i> is involved in polyphosphate metabolism

“…In eukaryotic organisms, polyP has been shown to be localized in various organelles, such as vacuoles (Allan and Miller, 1980), on the cell surfaces of yeasts (Tijssen et al, 1982), and in acidocalcisomes (Marchesini et al, 2002;Rodrigues et al, 2002;Ruiz et al, 2001a, b). It was reported that vacuolar polyphosphates are probably the natural substrate of alkaline phosphatase in Candida utilis (Fernandez et al, 1981).…”