The mitochondrial genome segregation system of T. brucei: Single p197 molecules connect the basal body with the outer membrane

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body of the flagellum. Here we studied the architecture of the exclusion zone filament of the TAC that connects the basal body with the mitochondrial outer membrane. The only known component of the exclusion zone filaments is p197. Using genetical, biochemical and microscopical methods we show that p197 has three domains all of which are essential for mitochondrial DNA inheritance. The C-terminus of p197 interacts with the mature and pro-basal body whereas its N-terminus binds to the peripheral outer membrane protein TAC65. The large central region of p197 has a high -helical content and likely acts as a flexible spacer. Replacement of endogenous p197 with a functional version containing N- and C-terminal epitope tags together with expansion microscopy demonstrates that p197 alone can bridge the approximately 170 nm gap between the basal body and the periphery of the outer membrane. This demonstrates the power of expansion microscopy which allows to localize distinct regions within the same molecule and suggests that p197 is the TAC subunit most proximal to the basal body. Significance statementSegregation of the replicated single unit mitochondrial genome of Trypanosoma brucei requires a large hardwired structure that connects the organellar DNA with the flagellar basal body. The cytosolic part of this structure consists of filaments made of single p197 molecules, a protein larger than 600 kDa. p197 has three domains all of which are essential for its function. The N-terminus of p197 is anchored to the peripheral outer membrane protein TAC65 whereas its C-terminus connects to the base of the basal body. The large central domain forms an -helix and consists of at least 26 repeats of 175 aa in length. It provides a flexible linker bridging the approximately 170 nm between the outer membrane and the basal body