Recognition of ubiquitin-conjugates by the 26S protease

Multiubiquitination is a key step leading to the selective degradation of abnormal polypeptides and many important regulatory proteins by a large multisubunit enzyme--the 26S protease. However, the mechanism by which ubiquitin-conjugates are recognized by this enzyme is unknown. I used a protein binding assay to identify a subunit (S5a) of the 26S complex that binds multiubiquitinated lysozyme. S5a associates with free multiubiquitin chains--a result consistent with the hypothesis that attachment of a multiubiquitin chain to a protein targets it for destruction by the 26S protease. In addition, the affinity of S5a for ubiquitin polymers increases with increasing chain length, providing a possible explanation for the preference of the 26S protease for substrates conjugated to longer multiubiquitin chains. The biochemical properties of S5a inspired a model in which repeated binding domains in the subunit confer the ability to select for multiubiquitin chains. In support of this idea, the cDNA for S5a encodes a protein containing repeated sequences. Addition of S5a to in vitro extracts specifically inhibits proteolysis of both artificial and natural substrates of the ubiquitin pathway. These data suggest that S5a will serve as a valuable reagent for assessing the involvement of ubiquitin-mediated proteolysis in a variety of cellular events.