Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62

Autophagy. 2018;14(2):347-358. doi: 10.1080/15548627.2017.1407889. Epub 2018 Jan 24.

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.

Keywords: BiFC; K33; SQSTM1/p62; autophagy; ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy / physiology
  • Cell Line
  • Fluorescence
  • HEK293 Cells
  • Humans
  • Molecular Imaging / methods*
  • Optical Imaging / methods*
  • Polyubiquitin / metabolism*
  • Sequestosome-1 Protein / metabolism*
  • Ubiquitination*

Substances

  • SQSTM1 protein, human
  • Sequestosome-1 Protein
  • Polyubiquitin

Grants and funding

This work was supported by the Japan Agency for Medical Research and Development [grant number 15AeK0109047h0002]; Japan Society for the Promotion of Science (JSPS) [grant number 16K15423], [grant number 25460946].