Rad23 Promotes the Targeting of Proteolytic Substrates to the Proteasome

A family of proteins that contain amino-terminal ubiquitin (Ub)-like (UbL) domains has been shown to play a role in proteolysis (12, 13, 20, 24, 33, 35). Rad23 is the best-characterized protein in this class, and Rad23 proteins of Saccharomyces cerevisiae and humans are biochemically similar (17, 18, 30, 35, 37). Rad23 binds DNA repair factors that play essential roles in nucleotide excision repair (NER) (29, 32, 37) and specifically interacts with Rad4 to facilitate the assembly of a DNA repair complex on UV-damaged DNA (19, 22). Extensive characterization of Rad23 proteins from yeast and humans showed that they promoted the assembly of DNA repair complexes (19, 22) and stimulated the activity of Rad4/XPC (36). We reported previously that the UbL domains in both yeast and human Rad23 could interact with catalytically active proteasomes (35). This interaction is important for DNA repair because removal of UbLR23 caused sensitivity to UV light (47), although this mutant retained the ability to bind Rad4 (P. Tongaonkar and K. Madura, unpublished results). However, a well-defined biochemical function for Rad23 was unavailable. The addition of purified Rad23 protein to a reconstituted ubiquitination reaction mixture effectively blocked the formation of multi-Ub chains on a test substrate histone, H2B (30, 41). In agreement with this result, we found that the overexpression of Rad23 inhibited the degradation of test substrates in vivo (30). Additionally, it has been shown that overexpression of Rad23 causes stabilization of Pds1 (8). The discovery that Rad23 can bind Ub (5, 7), multi-Ub chains (33, 48), and ubiquitinated proteins (30) provides a mechanistic basis for these findings. Rad23 contains two Ub-associated (UBA) domains (21, 43) that play an important role in regulating the abundance of the cell cycle regulator Pds1 (5, 8). Recent studies showed that the UBA domains in Rad23, as well as Ddi1 and Dsk2, could interact with Ub and multi-Ub chains (5, 7, 13, 33, 48). Interestingly, Dsk2 and Ddi1 also contain UbL domains, and double mutants (rad23Δ dsk2Δ and rad23Δ ddi1Δ) display growth defects that are not apparent in the single-mutant strains (6, 8). Similar to results from our studies with Rad23 (30, 35), the Dsk2 protein was recently reported to bind ubiquitinated proteins and the proteasome in yeast cells (13, 33). Collectively, these findings suggest that proteins that contain both UbL and UBA domains represent a novel class of proteolytic regulators that have similar and partially redundant functions. Rad23 has a role that overlaps with that of Rpn10 (26), a subunit in the proteasome that binds multi-Ub chains (44). Loss of both proteins (rad23Δ rpn10Δ) resulted in pleiotropic growth and proteolytic defects that included extreme sensitivity to cold temperatures and inability to grow in the presence of amino acid analog canavanine (26). In addition, ubiquitinated proteins accumulated in the double mutant, suggesting a failure in their delivery to the proteasome. In agreement with this conjecture, we found that ΔUbLrad23 (a mutant lacking the UbL domain) could bind ubiquitinated proteins but was unable to alleviate any of the defects of a rad23Δ rpn10Δ mutant, underscoring the importance of the Rad23-proteasome interaction. We report here that Rad23 can bind ubiquitinated cellular proteins. Significantly, this interaction required the UBA domains, since specific mutants were deficient in binding ubiquitinated cellular proteins. Because Rad23 mutants that lacked either UbLR23 or functional UBA domains failed to suppress the defects of rad23Δ rpn10Δ mutants, we conclude that the interactions with both ubiquitinated proteins and the proteasome are required for Rad23 function. High-level expression of Rad23 resulted in a dramatic increase in the levels of ubiquitinated proteins in the proteasome. Furthermore, the increased association between ubiquitinated proteins and the proteasome required Rpn10. These intriguing results are to our knowledge the first evidence that reveals the interaction between ubiquitinated proteins and the proteasome. Collectively, our results provide compelling support for a “shuttle factor hypothesis,” wherein Rad23 and other proteins that contain UbL and UBA domains, such as Dsk2 (6) and Ddi1 (8), regulate the translocation of proteolytic substrates to the proteasome. As a further test of this idea, we found that overexpression of GST-UbLR23 resulted in significant stabilization of a test substrate, suggesting that unregulated interaction between UbLR23 and the proteasome can interfere with the efficient delivery of proteolytic substrates by Rad23.