Cytosolic Trypanosoma cruzi nucleoside diphosphate kinase generates large granules that depend on its quaternary structure

PEREIRA, CA; REIGADA, CHANTAL; SAYE, MELISA; DI GIROLAMO, FABIO; MIRANDA, MARIANA

EXPERIMENTAL PARASITOLOGY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2014

0014-4894

1 Nucleoside diphosphate kinase (NDPK) is a key enzyme in the control of cellular 3 concentrations of nucleoside triphosphates, and has been shown to play important 4 roles in many cellular processes. In this work we investigated the subcellular 5 localization of the canonical NDPK1 from Trypanosoma cruzi (TcNDPK1), the 6 etiological agent Chagas´ Disease, and evaluated the effect of adding an additional 7 weak protein-protein interaction domain from the green fluorescent protein (GFP). 8 Immunofluorescence microscopy revealed that the enzyme from wild-type and 9 TcNDPK1 overexpressing parasites has a cytosolic distribution, being the signal more 10 intense around the nucleus. However, when TcNDPK1 was fused with dimeric GFP it 11 relocalizes in non-membrane bounded granules also located adjacent to the nucleus. 12 In addition, these granular structures were dependent on the quaternary structure of 13 TcNDPK1 and GFP since mutations in residues involved in their oligomerization 14 dramatically decrease the amount of granules. This phenomenon seems to be specific 15 for TcNDPK1 since other cytosolic hexameric enzyme from T. cruzi, such as the NADP+ 16 -linked glutamate dehydrogenase, was not affected by the fusion with GFP. In 17 addition, in parasites without GFP fusions granules could be observed in a 18 subpopulation of epimastigotes under metacyclogenesis and metacyclic 19 trypomastigotes. Organization into higher protein arrangements appears to be a 20 singular feature of canonical NDPKs; however the physiological function of such 21 structures requires further investigation.