Ubiquitin‐associated (UBA) domains in Rad23 bind ubiquitin and promote inhibition of multi‐ubiquitin chain assembly

Abstract

Rad23 is a DNA repair protein that promotes the assembly of the nucleotide excision repair complex. Rad23 can interact with the 26S proteasome through an N-terminal ubiquitin-like domain, and inhibits the assembly of substrate-linked multiubiquitin (multi-Ub) chains in vitro and in vivo. Significantly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiquitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) domains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substratelinked multi-Ub chains, although a mutant that lacked both UBA domains was significantly impaired. These studies suggest that the interaction with Ub is required for Rad23 activity, and that other UBA-containing proteins may have a similar function.

Cbl-b interacts with ubiquitinated proteins; differential functions of the UBA domains of c-Cbl and Cbl-b

“…In contrast, the UBA domains of proteins in the UbL-UBA family, such as Rad23 and Ddi1, have been demonstrated to play a role in the proteolytic pathway, although it is not clear whether these proteins act to promote or inhibit proteolysis (reviewed in Madura, 2002). One mechanism by which the UBA domains of Rad23 may affect proteolysis is by the inhibition of the multiubiquitination of proteins (Chen et al, 2001;Raasi and Pickart, 2003); a similar prevention of protein multiubiquitination by the UBA b may modulate its E3 activity. The ability of the UBA b to bind ubiquitin could also play a role in the targeting of the E3 activity of Cbl-b, as has been suggested for the p97 (Cdc48p) adaptor protein p47 -the UBA domain of p47 is necessary for the AAA ATPase p97 to reassemble mitotic Golgi (Meyer et al, 2002).…”

“…Like the UBA b , the UBA domains of Rad23a, UBA(1) and UBA(2), bind ubiquitin (Bertolaet et al, 2001a;Chen et al, 2001;Wilkinson et al, 2001;Rao and Sastry, 2002;Raasi and Pickart, 2003;Ryu et al, 2003). While they have a similar structure (Hofmann and Bucher, 1996;Dieckmann et al, 1998;Withers-Ward et al, 2000;Mueller and Feigon, 2002;Ryu et al, 2003), there is very little sequence homology between these UBA domains ( Figure 4a).…”

“…Subsequently, it was shown that the UBA domains of a number of proteins interact with ubiquitin (Bertolaet et al, 2001b;Chen et al, 2001;Wilkinson et al, 2001;Funakoshi et al, 2002;Meyer et al, 2002;Rao and Sastry, 2002;Saito et al, 2002;Ciani et al, 2003;Raasi and Pickart, 2003;Ryu et al, 2003;Chim et al, 2004). In addition to binding ubiquitin, UBA domains are capable of interacting with ubiquitin-like domains (Ryu et al, 2003) and the ubiquitin-like protein, NEDD8 (Tanaka et al, 2003).…”

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

“…Other studies, however, point to a negative role for Rad23-family proteins in proteasome function. Rad23-family proteins can block extension of ubiquitin chains (Ortolan et al, 2000;Chen et al, 2001), and hHR23 proteins can block proteasome degradation of certain substrates (Hiyama et al, 1999;Raasi and Pickart, 2003). MEFs derived from targeted deletion of the mouse orthologs of hHR23 (mHR23A and B) demonstrated defects in xeroderma pigmentosa group-C (XP-C) protein stabilization, consistent with a role for mHR23 proteins in stabilizing specific proteasome substrates (Ng et al, 2003).…”

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

“…Here we report a novel nonproteolytic function of the ubiquitin receptor dDsk2 in transcription regulation. dDsk2 belongs to the UBA/UbL family of extraproteasomal receptors (reviewed in refs 10,11), where the ubiquitin-associated (UBA) domain directly binds the ubiquitin moiety of ubiquitylated substrates 12,13 and the ubiquitin-like (UbL) domain mediates interaction with the proteasome 14 . dDsk2 is highly evolutionarily conserved from yeast to humans 10,15,16 .…”