Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

Significance A substrate-conjugated polyubiquitin chain is accepted as the “canonical” proteasomal degradation signal. Using a cellular (human and yeast) proteomic screen in the exclusive presence of nonpolymerizable ubiquitin, we show that a large group of proteins is degraded by the proteasome following monoubiquitination. The screen also unraveled polyubiquitin-dependent substrates, as they are stabilized in the presence of this ubiquitin mutant. Notably, monoubiquitination- and polyubiquitination-dependent substrates display distinct important characteristics. Monoubiquitinated proteins are of lower molecular mass and of lesser structural disorder. The two groups can be assigned to defined cellular pathways. Furthermore, some of the characteristics are confined to either human or yeast cells, suggesting that the mechanism of action/recognition of the ubiquitin system in the two organisms are different somehow. The “canonical” proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established—in both human and yeast cells—a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.

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