Structural And Functional Study Of The Human Mitochondrial Translocase TIM22 Complex

Mitochondria are crucial for numerous cellular processes,including energy metabolism,programmed cell death,biosynthesis of amino acids,protein quality control and degradation.Most mitochondrial proteins are synthesized as precursors in the cytosol and then imported into appropriate mitochondrial subcompartments by specific protein translocase complexes,including the translocase of the outer membrane complex(TOM complex),the sorting and assembly machinery(SAM complex),the mitochondrial import complex(MIM complex)the carrier translocase of the inner membrane complex(TIM22 complex)and the presequence translocase of the inner membrane complex(TIM23 complex).The TIM22 complex is responsible for the translocation and insertion of hydrophobic membrane proteins,including mitochondrial carrier proteins and translocase subunits(Tim17,Tim22 and Tim23).The human TIM22 complex contains at least six components: Tim22(hypothetical channel-forming protein),Tim29,Tim9,Tim10 a,Tim10b and AGK.Despite advances in our understanding of the function and pathophysiology of the TIM22 complex,structural characterization of the TIM22 complex and molecular mechanism of carrier protein translocation have been sparse.In this study,we focused on our attention on the structural and functional of the human TIM22 complex,three main progresses are listed below:1.We have acquired the human mitochondrial translocase TIM22 complex with good behavior and solubility via in vitro recombinant co-expression system In this study,we co-expressed all six known components of the human TIM22 complex in human embryonic kidney(HEK)293F cell lines using in vitro recombinant co-expression(p MLink)system.After Flag-tag affinity purification followed by gel filtration,the resulting TIM22 complex displayed good resolution behavior.Further mass spectrometry(MS)analysis confirmed the identity of all six components of the human TIM22 complex.2.We determined the structure of the human mitochondrial translocase TIM22 complex using single-particle cryo-EM Through detergent screening,p H buffer system screening,and cryo-electron microscope sample preparation conditions screening,we obtains optimal samples that can be used for data collection using 300 k V cryo-EM facility.For the complete data set of the human TIM22 complex,2,401,464 particles were auto-picked from 14,105 micrographs for 2D classification,3D classification,3D reconstruction,and model building and refinement.We determined the structure of the human TIM22 complex at an overall resolution of 3.73 angstrom and an intermembrane region resolution of 3.53 angstrom.3.Structural analysis and biochemical verification of the human mitochondrial translocase TIM22 complex.The human TIM22 complex structure contained one Tim22 molecule,one Tim29 molecule,one AGK molecular,and two hexamer chaperones,Tim9-Tim10a(3:3)and Tim9-Tim10a-Tim10b(2:3:1).The core subunit,Tim22,contains four transmembrane helices,forming a cavity that open to the lipid bilayer.We suggests that Tim22 might functioned similar to the Yid C insertase family.Tim29 is a single transmembrane protein that provides an N-terminal helix to stabilize Tim22 and a C-terminal intermembrane space(IMS)domain to connect AGK and two small TIM chaperone hexamers to maintain the TIM22 complex integrity.The results of BN-PAGE analysis and in vitro import assay suggested that the isolated TIM22 complex represents a functional complex.Mutagenesis analysis confirmed that the structure of the human TIM22 complex is correct.The present study revealed the assembly and detailed structural information of the TIM22 complex,and provided a structural basis for us to understand the molecular mechanism of the TIM22 complex in carrier protein transportation and insertion.It also provides a potential drug target for mitochondrial transport system related diseases.