Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform
Figure 2
TcADKn nuclear localization signal.
Deletion analysis and GFP fusions were built in order to determine the sequence responsible for TcADKn nuclear localization. The scheme on the left side shows the GFP fusions, indicating TcADKn amino acids range fused to GFP (Amino acids) and the resulting subcellular localization (Nuclear). T. cruzi epimastigotes were transfected with the constructions and fluorescence was followed by fluorescence microscopy NtADKn represents aminoterminal of the protein (1–89 amino acids), CtADKn represents the carboxiterminal of the protein (88–182 amino acids), NtNtADKn represents the aminoterminal of Nt (1–45 amino acids), Nt–Ct represents NtADKn were the p-loop has been deleted (1–9, 23–89 amino acids), CtNtADKn represents the carboxiterminal of NtADKn (40–89 amino acids). The non-classical NLS was mapped to the N-terminal (Nt) of the protein, being the catalytic lysine of the p-loop (K20) essential for its localization (ADKnK20R). Images corresponding to TcADKn, 4′,6-diamidino-2-phenylindole (DAPI), differential interference contrast microscopy (DIC) and the merged images, are showed in the right panel.