In cell biology, unraveling the complexities of cellular function at the molecular level remains a paramount endeavor. Significant scientific attention has been paid to understanding interactions at organelle contact sites, especially between mitochondria and the endoplasmic reticulum (ER). These sites are critical centers for the exchange of essential biomolecules, such as lipids and calcium, which are vital for maintaining cellular homeostasis. Disruptions in this communication between organelles are implicated in the development of various diseases, including neurodegenerative disorders, emphasizing the need to elucidate the mechanisms that govern organelle interactions. However, the study of these dynamic complexes presents significant challenges due to the lack of available tools, complicating the quest to understand the contact sites between the ER and mitochondria.
Following this need, a new strategy called “OrthoID” has been developed through the collaborative efforts of scientists from POSTECH, Daegu Catholic University College of Medicine, and Seoul National University. Featured in Nature CommunicationsOrthoID addresses this challenge by refining our ability to identify proteins that mediate these critical conversations.
Traditional methods relied heavily on the naturally derived streptavidin-biotin (SA-BT) binding system to label and isolate these mediator proteins. However, this approach has its limitations, particularly in capturing the full spectrum of protein interactions between two different organelles. OrthoID overcomes these limitations by introducing an additional synthetic binding pair, cucurbit.[7]uryl-adamantane (CB[7]-Ad), to work alongside SA-BT. The combination of mutually orthogonal binding pair systems allowed more precise identification and analysis of mediator proteins that freely translocate between the ER and mitochondria, facilitating deeper exploration of proteins involved in organelle contact sites. and discovering their roles in cellular functions and diseases. mechanisms.
Through meticulous experiments, researchers have demonstrated the effectiveness of OrthoID in quickly and accurately labeling proteins involved in dynamic organelle communication processes. By leveraging proximity labeling techniques (APEX2 and TurboID) with orthogonal binding pair systems, the method efficiently labeled and isolated proteins that facilitate critical interactions between mitochondria and the ER. This approach not only identifies known proteins involved in contacts between the ER and mitochondria, but also uncovers new candidate proteins, including LRC59, whose functions at the contact site were previously unknown. Furthermore, they also successfully identified the multiple sets of proteins that undergo structural and locational changes at the ER-mitochondria junction during critical cellular processes such as mitophagy, where damaged mitochondria are the target of degradation.
“OrthoID’s flexibility and modularity are among its greatest strengths.” says Professor Kimoon Kim, who led the POSTECH research. “This adaptability not only allows the study of various organelle contact sites, but also opens new avenues to explore complex cellular communications, overcoming the technical limitations of existing methods.”
Professor Kyeng Min Park from Daegu Catholic University School of Medicine adds: “OrthoID is a versatile and useful research tool aimed at decoding the complex language of cellular communication. It is expected to facilitate discoveries that will have profound implications for understand cellular health. , elucidate the mechanisms of the disease and promote the development of new therapeutic strategies.”
The collaborative team included Prof. Kimoon Kim and Dr. Ara Lee from the Department of Chemistry, Dr. Gihyun Sung from the Division of Advanced Materials Science at Pohang University of Science and Technology (POSTECH), Prof. Kyeng Min Park of the Catholic University of Daegu College of Medicine, Professor Hyun-Woo Rhee of the Department of Chemistry, and Professor Jong-Seo Kim of the College of Biological Sciences of Seoul National University.
This work was supported by the National Research Foundation of Korea (NRF) and the Institute of Basic Sciences (IBS).