Characterization of novel and essential kinetoplast-associated proteins in Trypanosoma brucei

The kinetoplast is one of the defining features of kinetoplastid protists and represents a unique concentration of mitochondrial DNA. This subcellular structure is a highly complex assembly of thousands of mutually catenated, circular DNA molecules as well as up to one hundred dedicated proteins. These components work in tandem to replicate and segregate the mitochondrial genome during cellular division, additionally coordinating with the basal body and flagellum through the tripartite attachment complex (TAC) superstructure. Here, we screened the MitoTag localization repository and identified a number of previously undescribed hypothetical proteins exhibiting putative signals within the kinetoplast of Trypanosoma brucei. Through endogenous tagging we verify their association with the kinetoplast or TAC. The essentiality for several of these kinetoplast proteins (KP) was assessed by RNAi knock-downs, revealing that the newly characterized KP56, KP84 and KP86 are indispensable for growth of the procyclic stage. Additionally, KP37, KP56, and KP84 displayed alterations in the abundance of maxicircles or minicircles, while the depletion of KP84 and KP86 resulted in cell cycle alternations. Pulldown assays using the endogenously V5-tagged cell lines identified novel interactors, which were additionally subjected to endogenous tagging for subcellular localization, revealing two additional proteins (KP45 and KP66) with dual localization to the kinetoplast and throughout the mitochondrial lumen. This work represents the most extensive identification of novel KPs to date and provides a methodological pipeline for the characterization of remaining KPs to further understand this intricate molecular structure.