No abstract

Mitochondria fulfill central functions in cellular energetics, metabolism and signaling. The outer membrane TOM40 complex imports virtually all mitochondrial proteins, however, its architecture and the molecular mechanisms of preprotein translocation are unknown. We mapped the active translocator with resolution down to single amino acid residues, discovering distinct transport paths for hydrophilic and hydrophobic preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel interior to recruit intermembrane space chaperones that guide the transfer of hydrophobic preproteins. The translocator possesses an intricate architecture with three Tom40 β-barrel channels sandwiched 2 between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex is in exchange with a dimeric isoform that is crucial for assembly of new TOM40 complexes. The dynamic coupling of α-helical receptors, β-barrel channels and chaperones generates a versatile machinery that manages transport of ~1,000 different proteins into mitochondria.One Sentence Summary: Architecture of the mitochondrial TOM40 entry gate identifies preprotein paths and the blueprint for its assembly.Main Text: Mitochondria are essential organelles in eukaryotic cells. They are pivotal for cellular ATP production, numerous metabolic pathways and regulatory processes, and programmed cell death. During evolution of eukaryotes, most genes for mitochondrial proteins were transferred to the nucleus. The proteins are synthesized as preproteins in the cytosol and imported back into mitochondria. Different classes of preproteins have been identified that either contain N-terminal targeting sequences (presequences) or internal targeting information in the mature part (1-3). The protein translocator of the outer membrane (TOM40 complex) functions as the main entry gate of mitochondria (1-3). Most of the >1,000 different mitochondrial proteins are imported by the TOM40 complex, followed by transfer to distinct intramitochondrial machineries specialized for individual classes of preproteins. Whereas the structurally known membrane protein complexes consist of either α-helical or β-barrel proteins, the TOM40 complex is composed of both α-helical and β-barrel integral membrane proteins. The complex consists of the channel-forming β-barrel protein Tom40 and six other subunits each containing single α-helical transmembrane (TM) segments: the receptor proteins Tom20, Tom22 and Tom70, and the small regulatory subunits Tom5, Tom6 and Tom7 (1-3). Tom40, Tom22 and the small Tom proteins form the TOM40 core complex, whereas Tom20 and Tom70 are more loosely associated with the complex. The molecular architecture of the complex has not been elucidated. It is thus unknown how α-helical and β-barrel membrane proteins can be combined into a functional complex and how diverse classes of preproteins can be transported by the same transmembrane channel.To define the archite...