Abstract / Summary
The mitochondrial proteomes of Leishmania tarentolae and Trypanosoma brucei contain ∼1,700 proteins, most of which lack homologs in higher eukaryotes. Here, we integrate complexome profiling and cryo-electron microscopy to define conserved and lineage-specific macromolecular assemblies that support mitochondrial functions in these kinetoplastid protozoa. Comparative analyses reveal species- and life-stage-dependent differences in the abundance and composition of respiratory, metabolic, RNA processing, and other complexes, refining and expanding current annotations. Structures of L. tarentolae respiratory complex III 2 (CIII 2 ), complex IV 2 (CIV 2 ), and complex V (CV) identify nine previously unrecognized nuclear-encoded subunits and resolve five mitochondrially encoded proteins, including products of pan-edited mRNAs. These reconstructions uncover architectural innovations: a subunit 8 of ubiquinol cytochrome-c reductase (QCR8) N-terminal extension that plugs the vestigial mitochondrial processing peptidase (MPP)α/β cavity in CIII 2 , a CIV 2 dimer stabilized by an extensive clade-restricted interface, and a CV dimer containing the mitochondrially encoded ATP6 subunit. Together, our findings reveal how kinetoplastids assemble specialized mitochondrial machinery while incorporating diverged components into core modules shared across eukaryotes.