Cytosolic Trypanosoma cruzi nucleoside diphosphate kinase generates large granules that depend on its quaternary structure.
Nucleoside diphosphate kinase (NDPK) is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in many cellular processes. In this work we investigated the subcellular localization of the canonical NDPK1 from Trypanosoma cruzi (TcNDPK1), the etiological agent Chagas's Disease, and evaluated the effect of adding an additional weak protein-protein interaction domain from the green fluorescent protein (GFP). Immunofluorescence microscopy revealed that the enzyme from wild-type and TcNDPK1 overexpressing parasites has a cytosolic distribution, being the signal more intense around the nucleus. However, when TcNDPK1 was fused with dimeric GFP it relocalizes in non-membrane bounded granules also located adjacent to the nucleus. In addition, these granular structures were dependent on the quaternary structure of TcNDPK1 and GFP since mutations in residues involved in their oligomerization dramatically decrease the amount of granules. This phenomenon seems to be specific for TcNDPK1 since other cytosolic hexameric enzyme from T. cruzi, such as the NADP(+)-linked glutamate dehydrogenase, was not affected by the fusion with GFP. In addition, in parasites without GFP fusions granules could be observed in a subpopulation of epimastigotes under metacyclogenesis and metacyclic trypomastigotes. Organization into higher protein arrangements appears to be a singular feature of canonical NDPKs; however the physiological function of such structures requires further investigation.
Laboratorio de Biología Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina.
Claudio A
CA
Pereira
Laboratorio de Biología Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina.
Chantal
C
Reigada
Laboratorio de Biología Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina.
Melisa
M
Sayé
Laboratorio de Biología Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina.
Fabio A
FA
Digirolamo
Laboratorio de Biología Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina. Electronic address: mirandamariana@gmail.com.
Mariana R
MR
Miranda
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D015153:Blotting, Western; D003594:Cytoplasmic Granules / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D004072:Digitonin; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D049408:Luminescent Agents; D051379:Mice; D008856:Microscopy, Fluorescence; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D014349:Trypanosoma cruzi / Q000201:enzymology*
D000913:Antibodies, Protozoan; D007202:Indicators and Reagents; D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; D009701:Nucleoside-Diphosphate Kinase; D004072:Digitonin
Granules; Nucleoside diphosphate kinase; Oligomerization; Protein arrangement; Trypanosoma cruzi