Long Li's group at School of Life Sciences, Peking University published a paper entitled "Molecular pathway of mitochondrial preprotein import through the Molecular pathway of mitochondrial preprotein import through the TOM-TIM23 supercomplex", published in Nature Structural & Molecular Biology. The study reports structural and biochemical results of the TOM-TIM23 supercomplex that mediates protein transport in mitochondria, reveals a new pathway of protein import through the TOM complex across the mitochondrial outer membrane, and redefines the core composition of the TIM23 transporter complex in the inner mitochondrial membrane.
As one of the most important organelles in eukaryotic cells, mitochondria play important roles in energy supply, intracellular metabolism, homeostasis and apoptosis. Mitochondria have a unique inner and outer bilayer lipid membrane structure, with more than 1,000 proteins distributed inside. 99% of mitochondrial proteins are encoded by cytosolic genes, synthesized in ribosomes in the cytoplasm, and transported across the membrane to the mitochondria to perform their functions. Therefore, the transportation of mitochondrial proteins is critical for maintaining and regulating mitochondrial activity, and mutations in related genes are closely associated with the development of many metabolic diseases and cancers in humans. Li Long's group has been working to explore the molecular mechanisms by which mitochondrial proteins enter mitochondria, and published work includes resolving the pathway by which mitochondrial membrane proteins enter the inner membrane via the TIM22 transport complex (Zhang Y. et al Cell Research 2021). The role of the TOM-TIM23 supercomplex in mitochondrial protein transport is even more important in this study. This supercomplex, assembled from dozens of protein subunits, spans the inner and outer mitochondrial bilayer membranes and controls the transport of more than 500 mitochondrial soluble and membrane proteins. Over the past 40 years, researchers have extensively and intensively studied the individual subunits of the TOM and TIM23 transport complexes. However, due to the highly dynamic nature of this supercomplex, the understanding of the central question of how these subunits assemble to form the protein transport pathway is still very limited, especially for the TIM23 complex in the endosomal membrane, where the composition of the core transport unit has been very unclear.
In this study, the authors first started with the assembly of a stable TOM-TIM23 supercomplex, explored various assembly schemes, and finally chose to fuse green fluorescent protein with mitochondrial protein as the transport substrate, and captured the intermediate state of the TOM-TIM23 supercomplex for transporting protein substrates in yeast cells. After in vitro purification, this intermediate state could still be stabilized for further structural biology and biochemistry studies. By single-particle analysis of this intermediate state by cryo-electron microscopy, the authors found that there is a direct interaction between the TOM complex located in the outer membrane and the TIM23 complex located in the inner membrane, linking the inner and outer mitochondrial membranes, which efficiently delivers the protein substrate from the outer mitochondrial membrane to the inner membrane (Fig. 1a). In particular, the TOM complex was resolved to a resolution of 4.1 Å. The structure revealed a "polar amino acid pathway" within the inner wall of the Tom40 channel subunit, which is conserved across species and mediates the passage of the protein substrate through the Tom40 intermediate pore in an unfolded form (Fig. 1b). This pathway is different from the two transmembrane pathways in Tom40 previously proposed based on biochemical experiments, demonstrating the versatility of the TOM complex in efficiently recognizing and transporting various mitochondrial proteins.
Fig. 1. Structure of the TOM-TIM23 supercomplex. (a) Schematic assembly and cryo-electron microscopy structure of the TOM-TIM23 supercomplex. (b) Cryo-electron microscopy structural model of the TOM complex containing the protein substrate. (c) AlphaFold2 structural model of the Tim17-Tim23-Mgr2 heterotrimer
In addition, the authors systematically introduced unnatural amino acids at different sites of the protein substrate and probed the surroundings of the protein substrate in the transport pathway of TOM-TIM23 by using photocrosslinking, thus identifying the core subunit that directly helps the substrate to cross the two lipid membranes. The experimental results showed that the C-terminal fragment of the protein substrate can cross-link with the Tom40 subunit of the outer membrane, a result consistent with structural observations. Surprisingly, however, the N-terminal fragment of the protein substrate cross-links with both Tim17 and Mgr2 subunits in the inner membrane, but not with the Tim23 subunit, which is commonly identified as constituting the inner membrane transport channel. This result suggests that there may be a significant bias in the perception of the core transport pathway of the TIM23 complex in past studies. To further identify the core transporter subunits and pathways in TIM23, the authors used a combination of AlphaFold2 modeling, biochemical cross-linking, yeast genetics, and in vitro transport of mitochondrial proteins, and found that the core component of the TIM23 complex is a heterotrimer consisting of Tim23, Tim17, and Mgr2 subunits, with Tim17 and Mgr2 face-to-face form a channel-like structure, while Tim23 binds to Tim17 in a back-to-back form and is not involved in channel formation (Fig. 1c). During protein translocation, the protein substrate passes through the channel-like structure formed by Tim17 and Mgr2 and does not directly contact the Tim23 subunit. Mutagenesis experiments revealed that knockdown of Mgr2 in yeast did not affect protein translocation, suggesting that only Tim17 is required in the channel-like structure formed by Tim17 and Mgr2.The reason why Tim17 can help proteins to cross the endosomal membrane is due to its unique surface amino acid distribution: on the one hand, Tim17 has a highly conserved negatively charged region at the entrance of the endosomal membrane that can disrupt the localized On the one hand, Tim17 has a highly conserved negatively charged region at the entrance of the inner membrane, which can disrupt the local phospholipid bilayer structure and lower the energy barrier for proteins to cross the lipid membrane; on the other hand, Tim17 has a conserved hydrophobic region at the center of the pathway, which helps to keep the inner membrane of the mitochondrion in an airtight condition and to maintain the membrane potential necessary for mitochondrial physiological activities. Taking together the results of various structural and biochemical experiments, it can be concluded that the TIM23 complex, with the Tim17 subunit at its core, adopts a mixed mode to help protein translocation, i.e., Tim17 can either dynamically bind with other subunits to form a channel to help proteins across the membrane, or Tim17 works in the insertion enzyme work mode to accomplish protein translocation alone without the need for channel formation. This result corrects the misperception in the field over the past 40 years that the Tim23 subunit constitutes a core channel.
In summary, this study developed a novel strategy for protein transport studies, revealed the critical pathway for mitochondrial proteins to cross the bilayer lipid membrane via the TOM-TIM23 supercomplex (Figure 2), overturned the long-standing field knowledge of the core components of the inner membrane TIM23 transport complex, and discovered a completely new mode of protein transport enzyme operation, which lays a solid foundation for a comprehensive and in-depth understanding of mitochondrial biosynthesis. synthesis, laying a solid foundation for a comprehensive and in-depth understanding of mitochondrial biosynthesis.
Figure 2. mitochondrial TOM-TIM23 transporter pathway
Long Li is the corresponding author of the paper, and Xueyin Zhou, a PhD student from the Peking University-Tsinghua Joint Center for Life Sciences class of 2018, Yuqi Yang, a PhD student from the School of Life Sciences class of 2019, Dr. Peng Wang from the Peking University cryo-electron microscopy platform, and Shanshan Wang, a PhD student from the School of Life Sciences class of 2020, are the co-first authors. Dongjie Sun, a former technician in Li Long's lab, Xiaomin Ou, a PhD student in the College of Life Sciences, class of 2022, and Yuke Lian, a PhD student in the College of Life Sciences, class of 2021, made significant contributions to this research. Prof. Ning Gao and Associate Researcher Ningning Li from School of Life Sciences, Peking University, and Researcher Xinzheng Zhang from Institute of Biophysics, Chinese Academy of Sciences, provided great help in cryo-electron microscopy data calculation, and Prof. Qing Li and Prof. Gain Chang from School of Life Sciences, Peking University, provided important guidance on yeast genetics and photocrosslinking experiments, respectively. The research work was supported by Peking University cryo-electron microscopy platform, High Performance Computing Center, Instrumentation Center of School of Life Sciences, and National Phoenix Protein Platform, and was funded by State Key Laboratory of Membrane Biology, Peking University-Tsinghua Joint Center for Life Sciences, and National Natural Science Foundation of China.
