The ubiquitin-proteasome system (UPS) orchestrates many cellular and tissue-specific processes by degrading damaged and key regulatory proteins. To enable investigation of UPS activity in different cell types in a living animal, we developed a photoconvertible fluorescent UPS reporter system for live imaging and quantification of protein degradation in Caenorhabditis elegans. Our reporter consists of the photoconvertible fluorescent protein Dendra2 targeted for proteasomal degradation by fusion to the UbG76V mutant form of ubiquitin. In contrast to previous reporters, this system permits quantification of UPS activity independently of protein synthesis. Our reporter revealed that UPS-mediated protein degradation varies in a cell type–specific and age-dependent manner in C. elegans.
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This study was supported by grants to C.I.H. from the Academy of Finland (113485 and 118450), the International Human Frontier Science Program Organization, Biocentrum Helsinki, the Sigrid Jusélius Foundation and the Magnus Ehrnrooth Foundation. G.H. was additionally supported by the University of Helsinki and O.M. by the Helsinki Biomedical Graduate School. We thank members of the Caenorhabditis Genetics Center for providing the N2 wild-type worms, S. Mitani (National Bioresource Project for the Nematode, Japan) for the rpn-10 mutant worms, R.I. Morimoto (Northwestern University) for the PF25B3.3::yfp expression vector and members of the Biomedicum Helsinki Molecular Imaging Unit for their help with confocal microscopy and imaging.
The authors declare no competing financial interests.
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Hamer, G., Matilainen, O. & Holmberg, C. A photoconvertible reporter of the ubiquitin-proteasome system in vivo. Nat Methods 7, 473–478 (2010) doi:10.1038/nmeth.1460
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