UBL-UBA Proteins Research Articles

Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain

Budding yeast Dsk2 is a family of UbL–UBA proteins that can interact with both polyubiquitin and the proteasome, and is thereby thought to function as a shuttle protein in the ubiquitin–proteasome pathway. Here we show that Dsk2 can homodimerize via its C-terminal UBA domain in the absence of ubiquitin. Dsk2 mutants defective in the UBA domain do not dimerize and do not bind polyubiquitin. The expression of Dsk2 UBA mutants fails to restore the growth defect caused by DSK2 disruption although that of wild-type Dsk2 can restore the defect. These results suggest that Dsk2 homodimerization via the UBA domain plays a role in regulating polyubiquitin binding in the ubiquitin–proteasome pathway.

The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease.

Ho endonuclease initiates a mating type switch by making a double-strand break at the mating type locus, MAT. Ho is marked by phosphorylation for rapid destruction by functions of the DNA damage response, MEC1, RAD9, and CHK1. Phosphorylated Ho is recruited for ubiquitylation via the SCF ubiquitin ligase complex by the F-box protein, Ufo1. Here we identify a further DNA damage-inducible protein, the UbL-UbA protein Ddi1, specifically required for Ho degradation. Ho interacts only with Ddi1; it does not interact with the other UbL-UbA proteins, Rad23 or Dsk2. Ho must be ubiquitylated to interact with Ddi1, and there is no interaction when Ho is produced in mec1 or Deltaufo1 mutants that do not support its degradation. Ddi1 binds the proteasome via its N-terminal ubiquitin-like domain (UbL) and interacts with ubiquitylated Ho via its ubiquitin-associated domain (UbA); both domains of Ddi1 are required for association of ubiquitylated Ho with the proteasome. Despite being a nuclear protein, Ho is exported to the cytoplasm for degradation. In the absence of Ddi1, ubiquitylated Ho is stabilized and accumulates in the cytoplasm. These results establish a role for Ddi1 in the degradation of a natural ubiquitylated substrate. The specific interaction between Ho and Ddi1 identifies an additional function associated with DNA damage involved in its degradation.

A post-ubiquitination role for MDM2 and hHR23A in the p53 degradation pathway.

Abrogation of ubiquitin/proteasome-dependent turnover of p53 is critical for its activation. UbL-UBA proteins, including human homolog of Rad23 (hHR23) proteins, may regulate proteasomal degradation of substrates such as p53, due to their ability to interact with both ubiquitinated substrates and the proteasome. siRNA-mediated depletion of hHR23A or hHR23B in human cell lines accelerated p53 degradation. In contrast, overexpression of hHR23 proteins led to the accumulation of ubiquitinated p53, and purified hHR23 proteins also blocked p53 proteasome degradation in vitro. An hHR23-MDM2 complex was identified, suggesting that MDM2 and hHR23 cooperate in the regulation of p53 proteasome degradation. Consistent with this hypothesis, an MDM2 mutant that demonstrated increased binding in vivo to hHR23A was able to ubiquitinate, but not degrade p53. Moreover, the defective phenotype of this MDM2 mutant was rescued by siRNA knockdown of hHR23A. Our data indicate that MDM2 acts at a step in the p53 degradation pathway after ubiquitination, to counteract hHR23 inhibition of p53 turnover. Moreover, our data suggest the possibility that ubiquitin ligase/UbL-UBA protein complexes, as exemplified by the MDM2/hHR23 complex, may serve a general role in regulating substrate degradation by the proteasome.

Rad23 Ubiquitin-associated Domains (UBA) Inhibit 26 S Proteasome-catalyzed Proteolysis by Sequestering Lysine 48-linked Polyubiquitin Chains

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.