线粒体TIM22复合体的冷冻电镜结构

人源线粒体TIM22复合体的冷冻电镜结构Liangbo Qi1, *, Qiang Wang1, *, Zeyuan Guan1, *, Yan Wu1, Jianbo Cao2, Xing Zhang3, Chuangye Yan4, #, and Ping Yin1, #1National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China.2Public Laboratory of Electron Microscopy, Huazhong Agricultural University, Wuhan, China3Department of Biophysics, and Department of Pathology of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Center of Cryo Electron Microscopy, Zhejiang University, Hangzhou, China.4Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China*These authors contributed equally to this work.#To whom correspondence should be addressed. E-mail: yinpingmail.hzau.edu.cn to Ping Yin or yancy2015mail.tsinghua.edu.cn to Chuangye YanAbstractMitochondria play vital functions in cellular metabolism and homeostasis. Most of the mitochondrial proteins are synthesized as precursors in the cytosol and imported into mitochondria for folding or maturation. The translocase TIM22 complex is responsible for the import of multiple hydrophobic carrier proteins to be folded in the inner membrane of mitochondria. In mammalian cells, the TIM22 complex consists of at least six components, Tim22, Tim29, AGK, and three Tim chaperons (Tim9, Tim10a and Tim10b). Here, we report the cryo-EM structure of human translocase TIM22 complex at 3.7-angstrom resolution. The core subunit, Tim22, contains four transmembrane helices, forming a partial pore that is open to the lipid bilayer. Tim29 is a single transmembrane protein, providing an N-terminal helix to stabilize Tim22 and a C-terminal intermembrane space (IMS) domain to connect AGK and two TIM chaperon hexamers to maintain the complex integrity. One TIM hexamer comprises Tim9 and Tim10a in 3:3 molar ratio, and the other consists of two Tim9, three Tim 10a, and one Tim10b. The latter hexamer faces the intramembrane region of Tim22, likely providing the dock to load the precursors to the partial pore of Tim22. Our structure serves as a molecular basis for mechanistic understanding of TIM22 complex function.IntroductionMitochondria are essential eukaryotic cells organelles with numerous functions such as energetics, metabolism, and cellular signaling etc. These functions are played by more than 1000 proteins. Although only a small set of which proteins are synthesized in mitochondria, most of proteins (99%) are encoded by nuclear genes, and imported into correct mitochondrial compartment by some specific translocase complexes. These translocation machineries are crucial for mitochondrial biogenesis, dynamics, and functions, which in turn are connected to dozens of mitochondrial diseases.One of protein import pathways, the carrier pathway, is mainly responsible for carrier proteins importing and inserting into the inner membrane. The carrier precursors pass through the pore of translocase of outer membrane (TOM complex), and then are transported by a hexameric small TIM chaperon complex (Tim9/Tim10a complex as a main form) to the carrier translocase, the translocase of inner membrane 22 complex (TIM22 complex). Consequently, the TIM22 complex mediates the insertion and lateral release of precursors into the inner membrane in a membrane potential-dependent manner.In human, the TIM22 complex consists of at least six components: a hypothetic channel-forming protein Tim22, three chaperons, (Tim9, Tim10a and Tim10b), newly identified Tim29 and acylglycerol kinase (AGK) (ref?) (fig. 1A). Tim22 and small chaperons are conserved from yeast to mammalian, but Tim29 and AGK are specific in metazoan (ref?). Despite advances in understanding of the functional aspects of TIM22 complex, structural characterization has been extremely slow. The limited structural information on TIM22 complex is Tim9/Tim10a, deduced from the yeast homologues, Tim9/Tim10, a hexamer structure (ref?). Here, we report the structure of human TIM22 complex at a nominal resolution of 3.7 , determined using single-particle cryoelectron microscopy (cryo-EM). The structure reveals the molecular architecture of the complex and provides an important framework for understanding the function of TIM22 complex in carrier protein maturation.ResultsI) Structure determination of the TIM22 complexWe co-expressed all of six components in human embryonic kidney (HEK) 293F cells. After flag-tag affinity purification followed by gel filtration, the resulting TIM22 complex displayed good solution behavior and were examined by Coomassie blue staining of SDSPAGE gels (fig. 1A). Mass spectrometric (MS) analysis of the purified complex confirmed the presence of all the components of TIM22 complex. Details of grid preparation, cryo-EM data acquisition, and structural determination of T