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

Abstract

We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl 2 and AgNO 3 . No glycerol transport was detected. Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H ؉ -PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.

Structural organization of Trypanosoma cruzi

“…This structure is formed by several tubules connected to a central vacuole located close to the flagellar pocket (Linder & Staehelin 1977). More recently, aquaporin, a protein involved in water transport, was identified in the epimastigotes of T. cruzi and shown to be localized to both acidocalcisomes and the contractile vacuole (Montalvetti et al 2004). These structures seem to be involved in process of osmoregulation.…”

A Cleavable Propeptide InfluencesToxoplasmaInfection by Facilitating the Trafficking and Secretion of the TgMIC2–M2AP Invasion Complex

“…TgM2AP⌬pro also overlapped with TgVP1, a "vacuolar" proton pyrophosphatase ( Figure 5D). Vacuolar proton pyrophosphatases acidify intracellular compartments, including digestive (Zhen et al, 1994) or contractile vacuoles (Montalvetti et al, 2004), acidocalcisomes (Montalvetti et al, 2004), or the trans-Golgi (Mitsuda et al, 2001) by using pyrophosphate hydrolysis to drive the translocation of protons across endomembranes.…”

Hyperosmotic Stress Induces Aquaporin-dependent Cell Shrinkage, Polyphosphate Synthesis, Amino Acid Accumulation, and Global Gene Expression Changes in Trypanosoma cruzi

“…In contrast to other aquaporins that are inhibited by high micromolar concentrations of either HgCl 2 or AgNO 3 , TcAQP1 was shown to be exquisitely sensitive to inhibition by very low concentrations of these compounds (16). To determine the importance of this aquaporin in water permeability during the hyperosmotic stress response, we treated cells with 1, 3, 5, and 10 M HgCl 2 , concentrations shown previously to inhibit osmotic swelling in Xenopus laevis oocytes transfected with TcAQP1 (16), and we then monitored volume changes during hyperosmotic stress using the light-scattering technique.…”

“…The epimastigotes transformed with pTEX-TcAQP-GFP constructs (16) were maintained in liver infusion tryptose medium supplemented with 5% heat-inactivated fetal bovine serum and 0.1-1 mg/ml geneticin (G418).…”

“…Aquaporin Mediates Water Efflux through Contractile Vacuole during Hyperosmotic Stress-In a previous study (16), we identified an aquaporin (TcAQP1) that is located in acidocalcisomes and the contractile vacuole complex of T. cruzi epimastigotes. In contrast to other aquaporins that are inhibited by high micromolar concentrations of either HgCl 2 or AgNO 3 , TcAQP1 was shown to be exquisitely sensitive to inhibition by very low concentrations of these compounds (16).…”