Identification and subcellular localization of TcHIP, a putative Golgi zDHHC palmitoyl transferase of Trypanosoma cruzi

Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex.

Confocal microscopy showing TcHIP immunolocalization at the Golgi complex of T. cruzi epimastigotes. Red: TcHIP; Green: TcRab7/GFP (Golgi marker); Merged image to the right: Blue–nucleus and kinetoplast; Yellow–TcHIP-TcRab7/GFP colocalization. Bar = 5 μm.Figure optionsDownload as PowerPoint slideHighlights
► TcHIP is a putative zDHHC palmitoyl transferase of T. cruzi.
► TcHIP is expressed in all developmental forms of Trypanosoma cruzi.
► TcHIP colocalized with TcRab7, a Golgi marker of T. cruzi.