1. Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62
- Author
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Masanori Kobayashi, Tetsuya Nakamura, Takashi Nagaishi, Shinichiro Nakada, Kiichiro Tsuchiya, Mamoru Watanabe, Hiroki Matsuda, Shigeru Oshima, Ryuichi Okamoto, Kana Otsubo, Emi Aonuma, Yoichi Nibe, Chiaki Maeyashiki, Yu Matsuzawa, and Yasuhiro Nemoto
- Subjects
0301 basic medicine ,macromolecular substances ,environment and public health ,Fluorescence ,Deubiquitinating enzyme ,Cell Line ,03 medical and health sciences ,Bimolecular fluorescence complementation ,Ubiquitin ,Sequestosome-1 Protein ,Autophagy ,Monoubiquitination ,Animals ,Humans ,Polyubiquitin ,Molecular Biology ,biology ,Optical Imaging ,Ubiquitination ,Colocalization ,Cell Biology ,Cell biology ,Molecular Imaging ,Complementation ,030104 developmental biology ,HEK293 Cells ,biology.protein ,Antibody ,Toolbox - Abstract
Ubiquitin chains are formed with 8 structurally and functionally distinct polymers. However, the functions of each polyubiquitin remain poorly understood. We developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay using Kusabira Green (KG) as a split fluorescent protein. The PolyUb-FC assay has the advantage that monoubiquitination is nonfluorescent and chain-specific polyubiquitination can be directly visualized in living cells without using antibodies. We applied the PolyUb-FC assay to examine K33-linked polyubiquitin. We demonstrated that SQSTM1/p62 puncta colocalized with K33-linked polyubiquitin and this interaction was modulated by the ZRANB1/TRABID-K29 and -K33 linkage-specific deubiquitinase (DUB). We further showed that the colocalization of K33-linked polyubiquitin and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) puncta was impaired by SQSTM1/p62 deficiency. Taken together, these findings provide novel insights into how atypical polyubiquitin is recruited by SQSTM1/p62. Finally, we developed an inducible-PolyUb-FC system for visualizing chain-specific polyubiquitin. The PolyUb-FC will be a useful tool for analyzing the dynamics of atypical polyubiquitin chain generation.
- Published
- 2018