Covalent modification of proteins is a common mechanism for alteration of protein function and stability. The small peptide ubiquitin is covalently linked to lysine residues on many proteins. Ubiquitination often targets proteins for degradation. The ubiquitin-proteasome system is responsible for quality control and regulatory functions in the cell. Existing methods for the detection of ubiquitination in a cell such as fluorescence resonance energy transfer and time-resolved fluorescence rely on donor-acceptor methods, and may be cumbersome and subject to interference by cellular components.
Researcher at the University of Michigan has developed an approach for the visualization of ubiquitinated proteins in living cells, designated ubiquitin-mediated fluorescence complementation (UbFC). This approach is based on complementation among fragments of fluorescent proteins when they are brought together by the covalent conjugation of ubiquitin fused to one fragment to a substrate protein fused to a complementary fragment. The UbFC strategy enables simultaneous visualization of proteins modified by different ubiquitin family peptides and comparison of their effects on protein localization. Visualization of ubiquitinated Jun revealed that it was localized predominantly to cytoplasmic structures. In contrast, Jun conjugated to small ubiquitin-related modifier 1 (SUMO1) was localized to subnuclear foci. Comparison of the distribution of ubiquitinated Jun with markers for various cytoplasmic compartments revealed that ubiquitinated Jun was localized to lysosomal vesicles. Fractionation of cell lysates confirmed that the majority of ubiquitinated Jun partitioned to the cytoplasmic fraction, and density gradient centrifugation analysis demonstrated that it co-sedimented with lysosomal beta-hexosaminidase activity. Mutation of a recognition sequence for the E3 ligase Itch/AIP4 prevented Jun ubiquitination and stabilized it in cells. Inhibition of lysosomal protein degradation by bafilomycin or chloroquine stabilized Jun but had no effect on the stability of mutated Jun that was not ubiquitinated by Itch/AIP4. The visualization of ubiquitinated Jun in living cells has uncovered a lysosomal pathway for Jun degradation that involves ubiquitination by Itch/AIP4.
Applications and Advantages
- Visualization of ubiquitinated proteins in living cells
- Enables simultaneous visualization of proteins modified by different ubiquitin family