| Summary: | The parasite Trypanosoma brucei is the causative agent of Human African trypanosomiasis and Nagana in cattle and millions of people in sub-Saharan Africa are at risk for both health and economic issues since current drugs are inadequate. N-linked glycosylation in Trypanosoma bruceidiffers from other eukaryotes and the parasite has several essential glycoproteins.Thus, the study of oligosaccharyltransferase activities is interesting for potential future drug discoveries. To assess whether TbSTT3A is essential in vitro(i.e. in culture), it was replaced by a drug resistance gene in the presence of a tetracycline inducible TbSTT3A ectopic copy. Although both endogenous TbSTT3A genes were replaced in the TbSTT3 locus, the TbSTT3A function was shown to be retained. These results might suggest that the cells have rearranged their genome during TbSTT3A replacement, which would imply that the tetracycline-inducible ectopic copy could not complement the loss of the endogenous TbSTT3A gene, suggesting that it is essential for cell viability in vitro. Additionally, in situ tagging of TbSTT3A enabled us to search for potential binding partners by performing blue native gel electrophoresis and immunoprecipitation using a Stable Isotope Labelling of Amino acids in Cell culture (SILAC) methodology. The results, further strengthened by co-immunoprecipitation, suggested that TbSTT3A forms a large multimeric complex with TbSTT3B in Trypanosoma brucei. To our knowledge, this is the first time two different STT3 enzymes have been reported to associate in an OST complex.
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