Revised Manuscript Received September 5, 1995® abstract: A necessary step in ubiquitin-dependent proteolysis is the addition of a polyubiquitin chain to the target protein. Thisubiquitinated protein is degradedby a multisubunit complex known as the 26S proteasome. The polyubiquitin chain is probably not released until a late stage in the proteolysis by the proteasome. It is subsequently disassembled to yield functional ubiquitin monomers. Here we present evidence that a 93 kDa protein, isopeptidase T, has the properties expected for the enzyme which disassembles these branched polyubiquitin chains. Protein and cDNA sequencing revealed that isopeptidase T is a member of the ubiquitin specific protease family (UBP). Isopeptidase T disassembles branched polyubiquitin chains (linked bythe G76—K48 isopeptide bond) by a sequential exo mechanism, starting at the proximal end of the chain (the proximal ubiquitin contains a free carboxyl-terminus). Isopeptidase T prefers to disassemble chains in which there is an intact and unblocked RGG sequence at the C-terminus of the proximal subunit. Rates of disassembly are reduced when G76 of the proximal ubiquitin is modified, for example, by ligation to substrate protein, by esterification, by replacement of the proximal glycine with alanine (G76A), or by truncation. Linearproubiquitin is only a poor substrate. Observed rates and specificity are consistent with isopeptidase T playing a major role in disassembly of polyubiquitin chains. The highdiscrimination against chains that are blocked or modified at the proximal end indicates that the enzyme acts after release of the chains from conjugated proteins or degradation intermediates. Thus, the proteolytic degradation signal is not disassembledby isopeptidase T before the ubiquitinated protein is degraded. These (and earlier) results suggest that UBP isozymes may exhibit significant substrate specificity, consistent with a role in the regulated catabolism of the polymeric ubiquitin, including the polyubiquitin protein degradation signal.

Ubiquitin-dependent proteolysis is a complex pathway of protein metabolism which has been implicated in a host of cellular functions, including maintenance of chromatin structure, ribosome biogenesis, cell cycle regulation, receptor function, oncoprotein degradation, the stress response, and degradation of abnormal proteins (Ciechanover & Schwartz, 1994; Jentsch, 1992a; Hershko & Ciechanover, 1992). Well over 50 enzymes are known to be involved in this pathway, and most are highly conserved between species. The molecular mechanism of ubiquitin action requiresits covalent attachment to proteins, including other molecules of ubiquitin. This post-translational ubiquitination1 is dependent on the formation of an isopeptide bond between the C-terminal glycine (G76) of ubiquitin and side chain amino groups of other ubiquitins or other proteins (Hershko & Ciechanover, 1992). The isopeptide bond is formed by the sequential action of three enzymes: El, the ubiquitin-activating enzyme; E2, one of a family of ubiquitin-