Ubiquitination-Induced Fluorescence Complementation (UiFC) for Detection of K48 Ubiquitin Chains In Vitro and in Live Cells

Zhiliang Chen,Shan Xu,Ilia V Baskakov,Yongwang Zhong,Yuxian Shen,Mariusz Karbowski,Zheng Liu,Shengyun Fang,Mervyn J Monteiro,Yang Wang,Maria G Masucci

doi:10.1371/journal.pone.0073482

Abstract

Proteins can be modified with eight homogenous ubiquitin chains linked by an isopeptide bond between the C-terminus of one ubiquitin and an amine from one of the seven lysines or the N-terminal methionine of the next ubiquitin. These topologically distinct ubiquitin chains signal for many essential cellular functions, such as protein degradation, cell cycle progression, DNA repair, and signal transduction. The lysine 48 (K48)-linked ubiquitin chain is one of the most abundant chains and a major proteasome-targeting signal in cells. Despite recent advancements in imaging linkage-specific polyubiquitin chains, no tool is available for imaging K48 chains in live cells. Here we report on a ubiquitination-induced fluorescence complementation (UiFC) assay for detecting K48 ubiquitin chains in vitro and in live cells. For this assay, two nonfluorescent fragments of a fluorescent protein were fused to the ubiquitin-interacting motifs (UIMs) of epsin1 protein. Upon simultaneous binding to a ubiquitin chain, the nonfluorescent fragments of the two fusion proteins are brought in close proximity to reconstitute fluorescence. When used in vitro, UiFC preferentially detected K48 ubiquitin chains with excellent signal-to-noise ratio. Time-lapse imaging revealed that UiFC is capable of monitoring increases in polyubiquitination induced by treatment with proteasome inhibitor, by agents that induce stress, and during mitophagy in live cells.

Highlights

Ubiquitin is a 76 amino acids long protein that is highly conserved in all eukaryotes
Bimolecular fluorescence complementation (BiFC) has been widely used for studying protein–protein interactions in live cells [33]. It is based on reconstitution of fluorescence of two nonfluorescent fragments of a fluorescent protein when they are brought in close proximity to each other by an interaction between proteins fused to the fragments
We investigated whether BiFC could be modified to monitor changes in ubiquitin chains by using ubiquitin-binding domains (UBDs) fused with nonfluorescent fragments of a fluorescent protein as the sensors

Summary

Introduction

Ubiquitin is a 76 amino acids long protein that is highly conserved in all eukaryotes. Cells use deubiquitinating enzymes (DUBs) to disassemble ubiquitin chains [2,7] These topologically distinct ubiquitin chains and their disassembly help govern remarkably functional diversity in ubiquitin signaling, such as targeting proteins for degradation, apoptosis, signal transduction, gene transcription, DNA repair, cell cycle progression, immune responses, virus budding, protein trafficking, and receptor and channel endocytosis [8,9]. Many of these functions control the life and death of cells. Aberrant ubiquitination has been widely associated with development of a wide range of devastating diseases, such as malignancies, inflammatory disorders, and neurodegenerative diseases [10]

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Conclusion