Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes.
Ubiquitin family peptide modifications regulate the functions and stabilities of many proteins. We have 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 cosedimented 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.
Fang
Deyu
D
Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0650, USA.
Kerppola
Tom K
TK
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D012097:Repressor Proteins; D025842:SUMO-1 Protein; D025801:Ubiquitin; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases