A frameshift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin (Ub), UBB(+1). UBB(+1) has been considered to inhibit proteasomes and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrate that expression of extended Ub variants leads to accumulation of heterogeneously linked polyubiquitin conjugates, indicating a pervasive effect on Ub-dependent turnover. 20S proteasomes selectively proteolyzed Ub extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of Lys48 linkages at 26S proteasomes. Processing of Lys48 and Lys63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of Ub-dependent degradation by extended Ub variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs.