p166 links membrane and intramitochondrial modules of the trypanosomal tripartite attachment complex

Abstract

ABSTRACTThe protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Thus, in contrast to the outer membrane TAC region which requires four essential subunits for proper function a single inner membrane TAC subunit is sufficient to bridge the distance from the OM to the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of non-essential TAC-associated proteins in the OM. These proteins can loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC but their binding would not be strong enough to withstand the mechanical force upon kDNA segregation.AUTHOR SUMMARYMitochondria evolved from a single endosymbiotic event and are a hallmark of eukaryotes. The large majority of genes for mitochondrial proteins are nuclear encoded now and only a small number are found in the mitochondrial genome. The protist Trypanosoma brucei is an extreme eukaryote in many aspects. For instance, trypanosomes have a single mitochondrion and its genome – called kinetoplast DNA (kDNA) – locates as a single unit inside the mitochondrion close to the basal body of the flagellum. The tripartite attachment complex (TAC) forms a connection between the basal body and the kDNA ensuring faithful segregation of kDNA among the daughter cells upon cytokinesis. Recently, several TAC subunits of the cytoplasm, the outer mitochondrial membrane (OM) and the mitochondrial matrix have been characterized. Here, we identify p166 as the first TAC subunit of the inner mitochondrial membrane. It is anchored with a single transmembrane domain separating the protein into a N-terminal moiety located in the matrix and a short C-tail. The latter reaches into the intermembrane space and binds the OM subunit TAC60 whereas the N-terminus interacts with the matrix subunit TAC102. Thus, with p166 we identified the missing link required to connect different modules of the TAC.