Bromodomain factor 1 of Trypanosoma cruzi is localized in the glycosomes, targeted by an N-terminal sequence

RITAGLIATI C; VILLANOVA VG; ALONSO VL; CRIBB P; SERRA EC

Mendoza

Congreso; XLVIII Reunion Anual Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2012

The bromodomain is the only known protein domain involved in the recognition of acetylated lysines. Lysine modification is a reversible and highly regulated posttranslational modification. New proteomics technology has enabled the identification of thousands of acetylated proteins distributed among the different compartments and involved in several processes. One of the most surprising findings has been that metabolic enzymes are highly represented among the acetylome. TcBDF1 contains a bromodomain in the N-terminal half of the protein. Western blot analysis of T. cruzi lysates with anti-TcBDF1, immunofluorescence microscopy of the different life cycle stages and immunoelectron microscopy of epimastigotes confirm TcBDF1´s non-nuclear localization. Co-localization assays with several markers suggest a glicosomal location. The amino acids sequence of TcBDF1 was analyzed with the PeroxisomeDB server, which recognized in its N-terminus a peroxisome-targeting signal type 2, one of the signals that direct glycosomal proteins into the matrix. To determine if the first 27 amino acids present in TcBDF1 are responsible of its import to the glycosome, we transiently transfected epimastigotes with constructs coding the whole protein, a truncated version which lacks the first 27 amino acids or only the N-terminus targeting signal, fused to the Red Fluorescent Protein. The intracellular localization of the different fusion proteins was determined by fluorescence microscopy. Our results confirm that BDF1 possesses a PTS-2 in its N-terminus, responsible of directing the protein to the glycosomes.