清大生醫學院分醫所廖品超助理教授（左一）與研究團隊成員

Assistant Professor Pin-Chao Liao of Institute of Molecular Medicine, National Tsing Hua University, with research team member.

論文標題Paper Title

Tom40 functions as a channel for protein retrotranslocation in the mitochondria-associated degradation (MAD) pathway.

學術期刊刊名Journal Name

Communications Biology

研究團隊 Research Team

主持人Principle Investigator：

廖品超 助理教授  Pin-Chao Liao, Assistant Professor

國立清華大學分子醫學研究所

Institute of Molecular Medicine, College of Life Science and Medicine (CLSM), National Tsing Hua University (NTHU).

參與者Participants:：

林茲瑩 Tzu-Ying Lin

國立清華大學分子醫學研究所

Institute of Molecular Medicine, College of Life Science and Medicine (CLSM), National Tsing Hua University (NTHU).

Catherine A. Tsang

哥倫比亞大學 病理與細胞生物學系

Department of Pathology and Cell Biology, Columbia University, New York, NY, United States

黃晨淨 Chen-Jing Huang

國立清華大學分子醫學研究所

Institute of Molecular Medicine, College of Life Science and Medicine (CLSM), National Tsing Hua University (NTHU).

Katherine Filipo

哥倫比亞大學 病理與細胞生物學系

Department of Pathology and Cell Biology, Columbia University, New York, NY, United States

Istvan Boldogh

哥倫比亞大學 病理與細胞生物學系

Department of Pathology and Cell Biology, Columbia University, New York, NY, United States

Liza A. Pon

哥倫比亞大學 病理與細胞生物學系

Department of Pathology and Cell Biology, Columbia University, New York, NY, United States

摘要

Abstract

粒線體為重要的細胞胞器，負責能量代謝、鈣離子恆定與氧化還原調控。為維持粒線體品質，細胞透過多種品質調控機制修復或清除受損的粒線體成分。其中，粒線體相關降解途徑（Mitochondria-associated degradation, MAD）可將粒線體蛋白質標記並送往蛋白酶體進行降解。本研究首次證實，位於粒線體基質中的MAD受質蛋白可利用ATP能量透過TOM通道跨越雙層膜進行逆向運輸。此發現揭示粒線體蛋白質移除與降解的關鍵機制，並增進我們對粒線體品質控制的理解。

Mitochondria are essential organelles that regulate energy metabolism, calcium homeostasis, and redox balance. To maintain mitochondrial integrity, cells employ multiple quality control pathways to repair or eliminate damaged components. Among these, mitochondria-associated degradation (MAD) selectively targets mitochondrial proteins for proteasomal degradation. In this study, we provide the first evidence that MAD substrates in the mitochondrial matrix can be retrotranslocated across both mitochondrial membranes through the TOM channel in an ATP-dependent manner. This finding uncovers a fundamental mechanism for mitochondrial protein turnover and advances our understanding of mitochondrial quality control.

研究成果Result/Contributions

粒線體相關降解途徑（mitochondria-associated degradation, MAD）透過泛素蛋白酶體系統（UPS），負責辨識並移除受損或錯誤摺疊的粒線體蛋白質，是維持粒線體蛋白品質的重要機制。過去研究已指出，MAD不僅作用於粒線體外膜蛋白，亦參與胞器內蛋白質的品質調控。然而，位於粒線體內部（如基質或內膜）的蛋白質，如何被運送至細胞質進行降解，其分子機制仍未明確。

本研究利用體外重構系統，成功建立MAD受質自粒線體基質跨越內外膜進行逆向運輸的實驗平台。結果顯示，此逆向轉運過程依賴ATP，但不依賴粒線體膜電位，顯示其機制不同於傳統的蛋白質運輸途徑。此外，本研究進一步探討粒線體外膜上的TOM複合體在此過程中的關鍵角色。當抑制Tom40通道時，MAD受質的逆向運輸顯著下降，證實Tom40為此過程的重要通道。

綜合上述結果，本研究支持一個新的模型：TOM複合體不僅作為蛋白質進入粒線體的通道，亦可作為雙向運輸系統，負責將受損蛋白質自粒線體內部逆向輸送至細胞質，進行後續降解。此一發現揭示粒線體蛋白質品質控制的關鍵步驟，並重新定義粒線體蛋白運輸系統在細胞品質控制中的功能與角色。

The mitochondria-associated degradation (MAD) pathway is a key mitochondrial quality control mechanism that mediates the recognition and elimination of damaged or misfolded mitochondrial proteins through the ubiquitin–proteasome system (UPS). Previous studies have established that MAD is essential for maintaining mitochondrial proteostasis and that its function extends beyond proteins localized to the mitochondrial outer membrane (MOM) to include proteins within the organelle. However, the molecular mechanism by which proteins residing inside mitochondria are removed and delivered to the cytosol for degradation has remained largely unclear.

In this study, we reconstitute the retrotranslocation of MAD substrates from the mitochondrial matrix across both the inner and outer mitochondrial membranes using cell-free systems. Our results demonstrate that this retrotranslocation process is dependent on ATP but independent of the mitochondrial membrane potential, indicating that it is mechanistically distinct from canonical protein import pathways.

Furthermore, we identify a critical role for the translocase of the outer membrane (TOM) complex in this process. The TOM complex, traditionally known as the primary entry gate for nuclear-encoded mitochondrial proteins, is shown here to also facilitate the export of proteins from mitochondria. Specifically, inhibition of protein translocation through the Tom40 channel significantly reduces the retrotranslocation of MAD substrates, demonstrating that Tom40 functions as a key channel for this pathway.

Together, our findings support a revised model in which the TOM complex acts as a bidirectional protein channel in the mitochondrial outer membrane. In addition to mediating protein import into the organelle, TOM also enables the retrotranslocation of damaged mitochondrial proteins to the cytosol for proteasomal degradation via the MAD pathway. This study provides the first direct evidence for a mechanistic link between mitochondrial protein quality control and cytosolic degradation systems, thereby advancing our understanding of how mitochondrial proteostasis is maintained at the molecular level.

論文連結 Paper Link

https://www.nature.com/articles/s42003-025-08549-z