The ubiquitin-proteasome system plays an essential role in the biology of eukaryotes. Through turnover of short-lived proteins, it regulates vital processes such as cell cycle progression, transcription, misfolded-protein degradation, and immune response. One scenario of how ubiquitinated proteins are driven to the 26S proteasome for degradation involves shuttle proteins (Rad23, Dsk2, or Ddi1), which recognize their substrate through a ubiquitin associated (UBA) domain and identify the proteasome through their ubiquitin like (UBL) domain.

Ddi1 (DNA Damage-Inducible 1) protein has an unusual composition for a UBL-UBA protein as it also contains a conserved retroviral protease fold domain (RVP). The detailed substrate specificity of Ddi1 as a shuttle is not known; however, it was found that Ddi1 is required for degradation of Ho endonuclease and F-box protein Ufo1, two proteins involved in cell cycle progression and regulation. In Saccharomyces cerevisiae, both UBA and UBL domains of Ddi1 are required for its shuttling function. Interestingly, through evolution, Ddi1 lost its UBA domain in mammals, which raises the question of how this shuttle protein performs its function without the domain that binds Ub. Furthermore, the presence of a UBL domain is also questionable since the N-terminal gene sequence of Ddi1 in yeast shares low identity with Ub and other known UBL domains. In order to fully confirm that yeast Ddi1 is an UBL-UBA shuttle, the solution structure of the nominal UBL domain was obtained. In addition, the functional properties of UBL domains were examined. This work shows that Ddi1UBL does not recognize its expected binding partners, one example being the ubiquitin interactive motif (UIM) domains of the Ufo1 protein. However, it is capable of recognizing ubiquitin (Ub) and its conjugates as well as the UBL domain of Dsk2, which is a novel interaction and is uncharacteristic for all known UBL domains.

To date, UBL-UBA shuttle proteins are well-studied proteins in the ubiquitin proteasomal pathway. Nevertheless, the details of target protein delivery to the proteasome for degradation are not well known. This work characterizes Ddi1 as an UBL-UBA protein, confirming its potential as a shuttle protein like other UBL-UBA proteins, such as Dsk2 and Rad23.