Multiple Interactions of Rad23 Suggest a Mechanism for Ubiquitylated Substrate Delivery Important in Proteolysis

Abstract

The mechanism underlying the delivery of ubiquitylated substrates to the proteasome is poorly understood. Rad23 is a putative adaptor molecule for this process because it interacts with ubiquitin chains through its ubiquitin-associated motifs (UBA) and with the proteasome through a ubiquitin-like element (UBL). Here, we demonstrate that the UBL motif of Rad23 also binds Ufd2, an E4 enzyme essential for ubiquitin chain assembly onto its substrates. Mutations in the UBL of Rad23 alter its interactions with Ufd2 and the proteasome, and impair its function in the UFD proteolytic pathway. Furthermore, Ufd2 and the proteasome subunit Rpn1 compete for the binding of Rad23, suggesting that Rad23 forms separate complexes with them. Importantly, we also find that the ability of other UBL/UBA proteins to associate with Ufd2 correlates with their differential involvement in the UFD pathway, suggesting that UBL-mediated interactions may contribute to the substrate specificity of these adaptors. We propose that the UBL motif, a protein-protein interaction module, may be used to facilitate coupling between substrate ubiquitylation and delivery, and to ensure the orderly handoff of the substrate from the ubiquitylation machinery to the proteasome.

A Novel Connexin43-interacting Protein, CIP75, Which Belongs to the UbL-UBA Protein Family, Regulates the Turnover of Connexin43

“…CIP75 may possibly interact with other cellular proteins, which in turn may affect the interaction of CIP75 with Cx43 and the proteasome. The UbL domain of Rad23 has been shown to directly interact with ufd2 (E4), and this interaction affects the UBA domain interaction with its target protein and the subsequent proteasome delivery and degradation (32). Thus, it is possible that other CIP75-interacting proteins may regulate the binding affinity of CIP75 with Cx43 and the proteasome, affecting its function in the proteasomal degradation process.…”

“…Members of the UbL-UBA domain-containing protein family have been implicated in different biological functions, such as nucleotide excision repair (29,30), spindle pole body duplication (31), and protein degradation (32)(33)(34). Notably, the Rad23 and PLIC2 proteins have been reported to interact with the S2/RPN1 or S5a/RPN10 components of the 19 S subunit of the 26 S proteasome complex via their UbL domains (30,35,36).…”

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

“…The handoff of ubiquitylated proteins from Cdc48 to the proteasome is facilitated by the interaction of Ufd2 with the UBL domains of Rad23 and Dsk2. Once dissociated from Ufd2, these UBL domains are free to deliver substrate to the proteasome (Richly et al 2005;Rumpf and Jentsch 2006;Hänzelmann et al 2010; see also Kim et al 2004).…”

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

“…For example, there are reports that UbL/UBA proteins may regulate substrate ubiquitination as well as proteasomal targeting. The UbL domain of Rad23 binds the ubiquitin ligase Ufd2, which cooperates with the chaperonelike AAA ATPase Cdc48 and its cofactors to ubiquitinate specific substrates (Richly et al, 2005;Kim et al, 2004;Schuberth et al, 2004). Moreover, when overexpressed, Rad23 and Dsk2 are capable of inhibiting proteolysis by binding a polyubiquitin chain on a substrate and inhibiting the ligation of additional ubiquitin molecules (Kleijnen et al, 2000;Ortolan et al, 2000).…”