Ddi1 is a ubiquitin-dependent protease

Abstract

TheSaccharomyces cerevisiaeprotein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.

Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

“…Intersects indicated in Figure 6A by arrows, are the subunits of the proteasome core, showing the strength of using mTPP in multiomics intersection analysis for identifying candidate protein subsets involved in the mechanism behind an observed phenotype. Aside from the core proteasome subunits, the additional member within this subset is the DNAdamage inducible protein 1 (Ddi1), a ubiquitin binding protein that functions as a proteasome shuttle (reviewed in (87)) that has recently been characterized as a ubiquitin-dependent protease (88). Ddi1 is destabilized in rpn5-ts as well (Supp.…”

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways

“…When cells are challenged with an excess of DPCs, they may require an alternative DPC protease. Ddi1 and 26S proteasome could at least partially substitute for Wss1 function (Serbyn et al, 2020), and both of them target ubiquitinated proteins for degradation (Finley et al, 2012;Yip et al, 2020). Ubiquitin and SUMO are known to compete for the acceptor lysines (Hendriks et al, 2014).…”

DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

“…Indeed, it would in many ways make sense in the context of the role of Ddi1/DDI2 as ubiquitin shuttles if infrequent cleavage of long ubiquitin chains facilitated the loading into the proteasome of a large variety of proteins with long ubiquitin chains. Interestingly, while this manuscript was in revision, Rapoport and co-workers reported complementary data that showed that the yeast Ddi1 protein preferentially (or exclusively) recognizes an artificial target protein when it carries a long ubiquitin chain and that it may even weakly cut the chain itself ( Yip et al., 2020 ). Importantly, our results clearly indicate that proteins with very long ubiquitin chains are slowly degraded by the UPS and require DDI2 activity for their timely removal in vivo .…”