Ubiquitin C-terminal Extension (CxUB) DUB cleavage assay

Ubiquitin is a conserved modifier regulating the stability and function of numerous target proteins. In all eukaryotes, polyubiquitin precursors are generated and processed into ubiquitin monomers. The final ubiquitin unit always contains a C-terminal extension, but its physiological significance is unknown. Here we show that C-terminally extended ubiquitin, termed CxUb, is essential for stress resistance, mitophagy and longevity in yeast and worms. CxUb forms ubiquitin chains and binds to a novel region within the ubiquitin chain-elongating E4 enzyme Ufd2, which also functions during stress and aging. Ufd2 recognizes CxUb and conjugates it to substrate proteins, triggering their degradation. In contrast, CxUb is not required for basal housekeeping functions of the ubiquitin-proteasome system. These data suggest that the C-terminally extended ubiquitin encodes a functionally unique ubiquitin form, specialized for proteostasis defects, expanding the code of post-translational modification processes.For the intact protein detection, please find the corresponding target proteins:1 NoDUb-T0 2 NoDUB-T103 NoDUB-T1204 UBP3ia-T105 UBP3ia-T1206 UBP3wt-T107 UBP3wt-T1208 UBP14-T109 UBP14-T12010 UBP15-T1011 Ubp15-T120