Structural Insights Into Assembly Of Human Mitochondrial Translocase TOM Complex

Mitochondria,containing approximately 1000-1500 proteins,plays important roles in cell energy transformation,metabolism,lipid biogenesis,signaling,and programmed cell death.Nearly 99%mitochondrial proteins are encoded by nuclear genes,and correctly transported into the mitochondrial subcompartment after translation in cytoplasm.Seven major membrane protein translocases complexes in mitochondria.TOM complex is the main entry gate for transferring protein precursors from the cytosol to the other mitochondrial translocase complexes.The TOM holo-complex consists of the channel-forming ?-barrel protein Tom40 and six other subunits,including the receptor proteins Tom20,Tom22,and Tom70 and three regulatory Tom proteins(Tom5,Tom6,Torn7),with each subunit containing single ?-helical transmembrane(TM)segments.In the absence of Tom70 and Tom20,the remaining five components constitutes a stable TOM core complex How ?-helical and ?-barrel membrane proteins assemble into a functional TOM complex remains to be further studied.In yeast,the TOM complex was characterized as two or three pores via electron microscope(EM)and crosslinking analyses,corresponding to the dimeric and the trimeric TOM complex respectively.The TOM c omplex could be dynamically converted between dimer and trimer,suggesting th at the TOM complex undergoes structural rearrangement during various stages of preprotein translocation across the outer membrane Each pore may regulate mutually to accomplish preprotein translocation independently or cooperatively,so as to be adapted for cotranslational import and highly efficient import.Most recently,high resolution cryo-EM structures of dimeric TOM core complex from Sacharomyces cerevisiae had been reported,and the TOM tetramer,equivalent to dimer of dimer,has also been observed,However,little is known about the structural information of trimeric TOM complex,More importantly,in human,the TOM complex is related to some mitochondrial diseases,such as Parkinson's disease(PD).In the damaged mitochondria,the TOM c omplex arrests PINK1 which then recruits and activates Parkin to induce mitophagy.F ailure to remove dysfunctional mitochondria might lead to early-onset PD development.Elucidation of dimeric and trimeric human TOM complex assembly could not only provide insights into the mechanism of protein translocation via TOM complex,but also lay a solid foundation for therapy of mitochondrial diseases.Here we presented the structures of human dimeric TOM core complex at 3.0 A resolution and the trimer conformation at 4.3 A resolution.The overall structure of the TOM core complex contains two copies of Tom40,Tom22,small Tom proteins,which form two symmetrical pores.Due to the low affinity of Tom70 and Tom20 with TOM core complex,we failed to obtain the structure of TOM hole complex containing these two receptors.The structure shows that ?1 and ?19 of Tom40 are buried at the dimer interface and stabilized by Tom22,making it difficult for Tom40 to open laterally.Based on the cryo-EM map,11 lipid-like EM densities were observed to encircle Tom40,and these lipids acted as a bridge to reinforce the assembly of a helix-P sheet.These provide structural basis for understanding the molecular transport mechanism of human TOM complex.Moreover,we analyze the structure of trimeric TOM complex and propose a possible dimer-trimer conversion model,which is consistent with the results from atomic force microscope and photo-crosslinking mass spectrometry investigation,suggesting that TOM complex can be existed in different oligomer states in the cell,thus facilitating substrate transportation.To verify that the trimeric TOM complex exists on human mitochondria,we used in situ cross-linking experiments to verify that the trimeric TOM complex we observed was correct.Relative to the dimeric TOM core complex,a structural rearrangement of Tom40 occurs in the trimeric TOM complex,making it possible to open the ? chain of one of Tom40 We speculate that the trimeric TOM complex may be adapted to the lateral release of Tom40.In summary,our data reveal the high-resolution structure of human dimeric TOM core complexes and the near-atomic-resolution structure of the human trimeric TOM complex.All of these findings provide a structural basis for understanding the molecular transport mechanism of the human TOM complex.We elucidated how lipids link the ?-helix and ?-sheet components.Moreover,we provided biochemical evidence of the trimeric TOM com plex in vivo,which suggests that the human TOM complex can exist in different oligomer states in a cell to facilitate substrate transport.For instance,in depolarized mitochondria,trimeric TOM might laterally release PINK1-like substrates into the outer mitochondrial membrane and lead to accumulation in the OMM.However,obtaining a high-quality cryo-EM map of the trimeric TOM complex to explain the roles of other TOM components in trimer assembly remains an unprecedented challenge Furthermore,obtaining the elaborate structures of the TOM complex with the precursor is of great importance for fu lly elucidating the mitochondrial protein translocation mechanism.