Assembly of the mitochondrial outer membrane module of the trypanosomal tripartite attachment complex

Abstract

The parasitic protozoan Trypanosoma brucei has a single mitochondrial nucleoid, anchored to the basal body of the flagellum via the tripartite attachment complex (TAC). The detergent-insoluble TAC is essential for mitochondrial genome segregation during cytokinesis. The TAC assembles de novo in a directed way from the probasal body towards the kDNA. However, the OM TAC module which is composed of five subunits, has previously been suspected to follow more complicated assembly pathways. Here, we identified four detergent-soluble OM TAC module subcomplexes that we assign to two classes. One class contains an oligomeric TAC40 complex that according to AlphaFold contains 6-8 subunits, as well as two subcomplexes of different sizes comprising TAC40, TAC42, and TAC60. The second class consists of a single complex composed of TAC65 and pATOM36. We show that the two subcomplex classes form independently and accumulate upon impairment of TAC assembly. The expression of an N-terminally truncated TAC60 variant causes the accumulation of the larger TAC40/TAC42/TAC60 complex and blocks completion of OM TAC module assembly. This suggests the following assembly pathway: i) TAC40 oligomerizes, ii) TAC42 and TAC60 bind the TAC40 oligomer forming two discrete larger intermediates, where iii) the larger subcomplex merges with the pATOM36/TAC65 subcomplex subsequently forming the OM TAC module. Author summaryMitochondria are essential for nearly all eukaryotes. This requires proper segregation of the organelles and their genomes to the daughter cells during cell division. For Trypanosomes and its relatives this represents a unique challenge because they have only a single highly compacted mitochondrial nucleoid, containing the bipartite mitochondrial genome, known as the kDNA. To ensure faithful segregation, kDNA replication is tightly coordinated with the nuclear cell cycle and the kDNA is physically connected to the flagellar basal body via the tripartite attachment complex (TAC), which spans both mitochondrial membranes. The TAC consists of at least nine subunits and its biogenesis arguably implies the most extreme lateral sorting event known for any mitochondrion. Five subunits, four integral and one peripheral outer membrane protein, make up the outer TAC module. Using blue native PAGE and RNAi analyses we have identified four soluble subcomplexes that likely represent assembly intermediates of the outer membrane TAC module. These results suggest that the outer membrane TAC module is assembled following a branched pathway that includes an unusual a ring-like intermediate of 6-8 subunits of a trypanosomatid-specific VDAC-like beta-barrel protein.