The UBA-UIM Domains of the USP25 Regulate the Enzyme Ubiquitination State and Modulate Substrate Recognition
Figure 2
Localization of the USP25m UBDs and analysis of their contribution to the deubiquitinating activity.
A. USP25m contains one UBA and two UIM (USP25_1, USP25_2) domains, as shown by alignments with other UBAs or UIMs. B. Schematic representation of the USP25m C-terminal and UBD deletion mutants: ΔUBA (Δ19-58 aa, inclusive), ΔUIM1 (Δ96-115 aa, inclusive), ΔUIM2 (Δ121-141 aa, inclusive), ΔUBA-UIM1 (Δ19-115 aa, inclusive), ΔUBA-UIM1-UIM2 (Δ19-141 aa, inclusive), ΔUIM1-UIM2 (Δ96-141 aa, inclusive). The constructs bearing serial deletions of USP25m at the C-terminus are also shown (E679X, E769X, Q863X, E1020X). C. Deubiquitinating activity assays indicated that UBDs were not required to cleave off ubiquitin (left upper panel). The mutant USP25mE679X was unable to hydrolyze Ub from the Ub-βgal substrate, indicating that the region between the amino acids 679 and 769 was required for enzymatic activity (right upper panel). The empty GST vector and the full length USP25m were respectively used as negative and positive controls. The expression level of each USP25m mutant was comparable (lower panels).