The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.

In bacteria with circular chromosomes, homologous recombination can generate chromosome dimers that cannot be segregated to daughter cells at cell division. Xer site-specific recombination at dif, a 28-bp site located in the replication terminus region of the chromosome, converts dimers to monomers through the sequential action of the XerC and XerD recombinases. Chromosome dimer resolution requires that dif is positioned correctly in the chromosome, and the activity of FtsK, a septum-located protein that coordinates cell division with chromosome segregation. Here, we show that cycles of XerC-mediated strand exchanges form and resolve Holliday junction intermediates back to substrate irrespective of whether conditions support a complete recombination reaction. The C-terminal domain of FtsK is sufficient to activate the exchange of the second pair of strands by XerD, allowing both intra-and intermolecular recombination reactions to go to completion. Proper positioning of dif in the chromosome and of FtsK at the septum is required to sense the multimeric state of newly replicated chromosomes and restrict complete Xer reactions to dimeric chromosomes.

The 26S proteasome degrades polyubiquitylated (polyUb) proteins by an ATP-dependent mechanism. Here we show that binding of model polyUb substrates to the 19S regulator of mammalian and yeast 26S proteasomes enhances the peptidase activities of the 20S proteasome about two-fold in a process requiring ATP hydrolysis. Monoubiquitylated proteins or tetraubiquitin alone exert no effect. However, 26S proteasomes from the yeast alpha3DeltaN open-gate mutant and the rpt2YA and rpt5YA mutants with impaired gating can still be activated (approximately 1.3-fold to 1.8-fold) by polyUb-protein binding. Thus, binding of polyUb substrates to the 19S regulator stabilizes gate opening of the 20S proteasome and induces conformational changes of the 20S proteasome that facilitate channeling of substrates and their access to active sites. In consequence, polyUb substrates will allosterically stimulate their own degradation.

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