Modification by single ubiquitin moieties rather than polyubiquitination is sufficient for proteasomal processing of the p105 NF-κB precursor

Activation of NF-κB is regulated via numerous ubiquitin- and proteasome-mediated steps - an important one is processing of the precursor p105 to the p50 active subunit. The mechanisms involved are largely unknown, as this is an exceptional case where the ubiquitin system does not destroy its substrate completely. Here we demonstrate that proteasomal processing of p105 requires ubiquitin, but not generation of polyubiquitin chains. In vitro, ubiquitin species that cannot polymerize mediate processing. In yeasts that express non-polymerizable ubiquitins, processing proceeds normally, whereas degradation of substrates that are dependent on polyubiquitination is inhibited. Similar results were obtained in mammalian cells. Interestingly, processing requires multiple monoubiquitinations, as progressive elimination of lysines in p105 is accompanied by gradual inhibition of p50 generation. Last, the proteasome recognizes the multiply monoubiquitinated p105. These findings suggest that a proteolytic signal can be comprised of a cluster of single ubiquitins, and not necessarily of a chain.