Sumoylation is a post-translational modification essential in most eukaryotes 
that regulates stability, localization, activity, or interaction of a multitude 
of proteins. It is a reversible process wherein counteracting ligases and 
proteases, respectively, mediate the conjugation and deconjugation of SUMO 
molecules to/from target proteins. Apart from attachment of single SUMO moieties 
to targets, formation of poly-SUMO chains occurs by the attachment of additional 
SUMO molecules to lysine residues in the N-terminal extensions of SUMO. In 
Saccharomyces cerevisiae there are apparently only two SUMO(Smt3)-specific 
proteases: Ulp1 and Ulp2. Ulp2 has been shown to be important for the control of 
poly-SUMO conjugates in cells and to dismantle SUMO chains in vitro, but the 
mechanism by which it acts remains to be elucidated. Applying an in vitro 
approach, we found that Ulp2 acts sequentially rather than stochastically, 
processing substrate-linked poly-SUMO chains from their distal ends down to two 
linked SUMO moieties. Furthermore, three linked SUMO units turned out to be the 
minimum length of a substrate-linked chain required for efficient binding to and 
processing by Ulp2. Our data suggest that Ulp2 disassembles SUMO chains by 
removing one SUMO moiety at a time from their ends (exo mechanism). Apparently, 
Ulp2 recognizes surfaces at or near the N terminus of the distal SUMO moiety, as 
attachments to this end significantly reduce cleavage efficiency. Our studies 
suggest that Ulp2 controls the dynamic range of SUMO chain lengths by trimming 
them from the distal ends.