In eukaryotes, ubiquitin (Ub)-dependent proteolysis is essential for bulk protein turnover as well as diverse processes including cell-cycle control, differentiation, antigen presentation, and the stress response. Generally, multiple ubiquitins are added onto a substrate to form an isopeptide-linked 'polyubiquitin' chain, which targets substrates for degradation by the 26S proteasome. The specificity of Ub-dependent degradation was thought to depend primarily on the selection of targets for ubiquitination, but recently we have reported evidence for a second level of specificity in which (poly)Ub-protein conjugates are partitioned among two fates: degradation of the protein substrate by the 26S proteasome; and disassembly by Ub isopeptidase(s) to regenerate the protein substrate. Potentially, an isopeptidase could influence degradation by 'editing' (poly)Ub-protein conjugates according to the extent of ubiquitination rather than the structure of the ubiquitination target itself. Here we describe a bovine isopeptidase that is well suited to such an editing function because of its unique localization and specificity. This enzyme is an intrinsic subunit of PA700, the 19S regulatory complex of the 26S proteasome. By disassembling the degradation signal from only the distal end of (poly)Ub chains, this isopeptidase can selectively rescue poorly ubiquitinated or slowly degraded Ub-protein conjugates from proteolysis.

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.

Alzheimer's disease is the most common cause of dementia in the elderly. Although several genetic defects have been identified in patients with a family history of this disease, the majority of cases involve individuals with no known genetic predisposition. A mutant form of ubiquitin, termed Ub ؉1 , has been selectively observed in the brains of Alzheimer's patients, including those with nonfamilial Alzheimer's disease, but it has been unclear why Ub ؉1 expression should be deleterious. Here we show that Ub ؉1 is an efficient substrate for polyubiquitination in vitro and in transfected human cells. The resulting polyubiquitin chains are refractory to disassembly by deubiquitinating enzymes and potently inhibit the degradation of a polyubiquitinated substrate by purified 26S proteasomes. Thus, expression of Ub ؉1 in aging brain could result in dominant inhibition of the Ub-proteasome system, leading to neuropathologic consequences.