A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi

J Biol Chem. 2004 Sep 10;279(37):38673-82. doi: 10.1074/jbc.M406304200. Epub 2004 Jul 12.

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). glycerol transport was detected. No 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry
  • Amino Acid Sequence
  • Animals
  • Aquaporins / biosynthesis
  • Aquaporins / chemistry*
  • Aquaporins / genetics
  • Asparagine / chemistry
  • Blotting, Northern
  • Blotting, Southern
  • Blotting, Western
  • Cloning, Molecular
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Glycerol / metabolism
  • Green Fluorescent Proteins
  • Immunoblotting
  • Immunohistochemistry
  • Luminescent Proteins / metabolism
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Oocytes / metabolism
  • Osmosis
  • Peptides / chemistry
  • Phylogeny
  • Plasmids / metabolism
  • Proline / chemistry
  • Protein Structure, Tertiary
  • Protons
  • Pyrophosphatases / chemistry*
  • Saccharomyces cerevisiae / metabolism
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Time Factors
  • Transfection
  • Trypanosoma cruzi / metabolism*
  • Trypanosoma cruzi / ultrastructure
  • Vacuoles / ultrastructure
  • Xenopus laevis

Substances

  • Aquaporins
  • Luminescent Proteins
  • Peptides
  • Protons
  • Green Fluorescent Proteins
  • Asparagine
  • Proline
  • Pyrophosphatases
  • Alanine
  • Glycerol

Associated data

  • GENBANK/AF312691