Role of Trypanosoma brucei Tim17 during cell survival and cell cycle progression

Abstract

Mitochondrial protein translocation machineries in the kinetoplastid parasites, like Trypanosoma brucei have poorly been characterized. BLAST analysis for protein translocases of mitochondrial outer and inner membrane (Tom and Tim) in the recently completed genome database of T. brucei only showed homologoues of few Tim proteins. The ORF for the putative TbTim17 encodes a protein of predicted molecular mass 16.2 kDa and showed 20–25% identity with other Tim 17. TbTim17 possesses the characteristic 4 transmembrane domains found in previously characterized Tim17. Phylogenetic analysis revealed that Tim17 from the kinetoplastid parasites are quite divergent than that from other eukaryotes. RNAi knockdown revealed that the gene is essential for cell survival. Within 48hrs after induction of RNAi, TbTim17 protein level was decreased to 70%, which resulted in a cessation of cell growth and changes in cell morphology. During this period, the elongated trypomastigotes were transformed to spherical forms with a single detached flagella. Electron microscopy and in situ fluorescent staining with DAPI revealed that TbTim17 RNAi cells contain large masses of DNA, which constitutes of multiple nuclei but a single kinetoplast. Flow cytometric analysis also showed that the cells were arrested at G2/M phase due to the reduction of TbTim17. After 48hrs of induction of RNAi, the expression level of several mitochondrial proteins were reduced 40 −90% such as MP81, MP63, REL1, MP42, Pol β PAK and HSP70. However, the steady state level of TAO remains unaltered, suggesting that the import of TAO is not dependent on Tim17. Together, these results suggest that the decrease of Tim17p inhibits kinetoplast segregation and thus blocked cytokinesis, however, mitosis remained uninhibited. This project is supported by NIH 3S06GM080837–3051