期刊
AUTOPHAGY
卷 14, 期 2, 页码 347-358出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/15548627.2017.1407889
关键词
autophagy; BiFC; K33; SQSTM1/p62; ubiquitin
类别
资金
- Japan Agency for Medical Research and Development [15AeK0109047h0002]
- Japan Society for the Promotion of Science (JSPS) [16K15423, 25460946]
- Grants-in-Aid for Scientific Research [15H04808, 25460946, 16H05286, 16K15423, 16H05284] Funding Source: KAKEN
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.
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