Characterization of the Trimeric TOM Complex by HS-AFM Single-Molecule Analysis.

The translocase of the outer mitochondrial membrane (TOM) complex is the entry gate for mitochondrial proteins synthesized in the cytosol. Previous biochemical studies indicated that the TOM complex functions as a trimer and exists in a dynamic equilibrium with a Tom22-lacking dimer in intact mitochondria. In contrast, recent cryo-electron microscopy (cryo-EM) studies revealed near-atomic structures of the Tom22-containing core dimer. However, the relationship between the core dimer and the functional trimer remains unclear. Here, we analyzed the oligomeric organization and structural properties of purified yeast TOM complexes using high-speed atomic force microscopy (HS-AFM). Purified yeast TOM complexes predominantly adopted a trimeric organization but underwent deformation and dissociation during repeated HS-AFM scanning, leaving dimeric and monomeric particles. Trimeric particles exhibited spherical shapes consistent with previous crosslinking models, whereas dissociated dimers closely resembled TOM core-dimer structures determined by cryo-EM. HS-AFM also revealed the peripheral localization of the Tom20 receptor and enabled direct visualization of substrate-TOM interactions, with TOM association accompanied by reduced substrate mobility. Together, these findings provide quantitative and structural evidence for a predominantly trimeric organization of the purified TOM complex and suggest that the trimer contains a stable core-dimer unit with a more loosely associated third Tom40-containing unit.