In-situ cross-linking mass spectrometry reveals compartment-specific proteasomal interactions and structural heterogeneity

Lili Zhao, Runtao Zhao, Zhou Gong, Fuxiang Liang, Nan Zhao, Bowen Zhong, Maili Liu, Yukui Zhang, Qun Zhao (), Lihua Zhang () and Chun Tang ()
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Lili Zhao: Chinese Academy of Sciences, State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics
Runtao Zhao: Peking University, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering
Zhou Gong: Chinese Academy of Sciences, State Key Laboratory of Magnetic Resonance Spectroscopy and Imaging, Innovation Academy for Precision Measurement Science and Technology
Fuxiang Liang: Zhejiang University, Department of Thoracic Surgery, the Second Affiliated Hospital, School of Medicine
Nan Zhao: Chinese Academy of Sciences, State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics
Bowen Zhong: Chinese Academy of Sciences, State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics
Maili Liu: Chinese Academy of Sciences, State Key Laboratory of Magnetic Resonance Spectroscopy and Imaging, Innovation Academy for Precision Measurement Science and Technology
Yukui Zhang: Chinese Academy of Sciences, State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics
Qun Zhao: Chinese Academy of Sciences, State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics
Lihua Zhang: Chinese Academy of Sciences, State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics
Chun Tang: Peking University, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering

Nature Communications, 2025, vol. 16, issue 1, 1-16

Abstract: Abstract The proteasome is an essential cellular machine that degrades ubiquitinated substrate proteins to maintain proteostasis. Studies of purified proteasomes, however, cannot fully recapitulate its complexity. Here, we employ in-situ cross-linking mass spectrometry (XL-MS) combined with nuclear-cytoplasmic fractionation to characterize human 26S proteasome within intact cells. Our analysis reveals extensive compositional and conformational heterogeneity between subcellular compartments, along with distinct interactomes and dynamic states. Notably, we identify additional ubiquitin-associated proteasomal subunits and uncover compartment-specific ubiquitin-binding and ubiquitination patterns. Further we identify previously unreported proteasome-interacting proteins, including deubiquitinase USP15, and reveal a hybrid proteasome variant wherein translation initiation factor EIF3M substitutes for subunit Rpn9. Leveraging cross-linking-derived distance restraints, we model transient interactions and dynamic subcomplexes of the proteasome. Together, our work establishes a robust framework for in-situ structural analysis of large protein complexes and provides mechanistic insights into how compartment-specific architectures and interactions regulate proteasome function.

Date: 2025
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DOI: 10.1038/s41467-025-65752-6

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