Ubiquitin-specific protease (USP) 25, belonging to the USP family of 
deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a 
~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of 
the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds 
to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a 
critical and cooperative role. Purified USP25 exhibited higher ubiquitin 
isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. 
Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs 
resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a 
role for the UIMs in exerting the full catalytic activity of USP25. Moreover, 
when mutations that convert the binding preference from Lys48- to Lys63-linked 
ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants 
acquired elevated and reduced isopeptidase activity toward Lys63- and 
Lys48-linked ubiquitin chains, respectively. These results suggested that the 
binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains 
contributes not only to the full catalytic activity but also to the ubiquitin 
chain substrate preference of USP25, possibly by selectively holding the 
Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core.