METABOLISM OF THE POLYUBIQUITIN DEGRADATION SIGNAL - STRUCTURE, MECHANISM, AND ROLE OF ISOPEPTIDASE-T

A necessary step in ubiquitin-dependent proteolysis is the addition of a polyubiquitin chain to the target protein. This ubiquitinated prote in is degraded by 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 en zyme which disassembles these branched polyubiquitin chains. Protein a nd cDNA sequencing revealed that isopeptidase T is a member of the ubi quitin specific protease family (UBP). Isopeptidase T disassembles bra nched polyubiquitin chains (linked by the 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). Isopeptida se T prefers to disassemble chains in which there is an intact and unb locked RGG sequence at the C-terminus of the proximal subunit. Rates o f disassembly are reduced when G76 of the proximal ubiquitin is modifi ed, for example, by ligation to substrate protein, by esterification, by replacement of the proximal glycine with alanine (G76A), or by trun cation. Linear proubiquitin is only a poor substrate. Observed rates a nd specificity are consistent with isopeptidase T playing a major role in disassembly of polyubiquitin chains. The high discrimination again st chains that are blocked or modified at the proximal end indicates t hat the enzyme acts after release of the chains from conjugated protei ns or degradation intermediates. Thus, the proteolytic degradation sig nal is not disassembled by isopeptidase T before the ubiquitinated pro tein is degraded. These (and earlier) results suggest that UBP isozyme s may exhibit significant substrate specificity, consistent with a rol e in the regulated catabolism of the polymeric ubiquitin, including th e polyubiquitin protein degradation signal.