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Summary Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine48-linked ubiquitin is essential for marking targets for proteasomal degradation but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine48 and lysine63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine48-linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin.

Summary As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone, and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin-chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly-tagged cargo. We propose a mechanism for a malleable ubiquitin-signal that depends both on chain-length and combination of receptors to produce tetra-ubiquitin as an efficient signal threshold.

The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for 1 H, 15 N, and 13 C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2's interactions with proteasomal receptors and its role as a polyubiquitin shuttle in the ubiquitin-dependent proteasomal degradation as well as other cellular pathways. KeywordsDsk2p; ubiquitin-like domain; UBL; proteasome Biological contextDsk2p, a yeast protein comprising a N-terminal ubiquitin-like (UBL) domain, two stressinduced phosphoprotein 1 (STI1) domains, and a C-terminal ubiquitin-associated (UBA) domain, was first isolated from Saccharomyces cerevisiae as a suppressor of kar1 allele defective for spindle pole body duplication (Biggins et al. 1996;Funakoshi et al. 2002). Dsk2p belongs to a class of UBL-UBA proteins proposed to act as polyubiquitin shuttles in ubiquitinmediated protein degradation, the principal mechanism for the turnover of short-lived proteins in eukaryotes. Characteristic for the modular composition of these proteins, which include Rad23 and Dsk2 families, is the presence of a UBL domain at or near the N-terminus and a UBA domain at the C-terminus. The bi-functional nature of these proteins is based on the ability of the UBL domain to bind to the proteasome (Funakoshi et al. 2002) while the UBA domain can bind monomeric ubiquitin (monoUb) and polyubiquitin (polyUb) chains (Wilkinson et al. 2001).Dsk2p appears to play a similar role to Rad23, another UBL-UBA protein in yeast. Both proteins have been found to mediate the interaction between polyubiquitinated substrates and the proteasome (Funakoshi et al. 2002;Elsasser et al. 2004;Fujiwara et al. 2004;Verma et al. 2004;Ghaboosi and Deshaies 2007). However, Dsk2p is distinct from Rad23 that contains two UBA domains. In hHR23a, the human homologue of Rad23, the C-terminal UBA-2 has a rather low affinity for monoUb, but binds strongly and selectively to Lys48-linked polyUb chains (Varadan et al. 2004;Raasi et al. 2005;Varadan et al. 2005). Dsk2p's UBA, on the other hand, binds strongly already to monoUb and appears to bind polyUb chains nonselectively (Ohno et al. 2005;Raasi et al. 2005;Zhang et al. 2008). The UBL domains of both Dsk2p and Rad23 bind proteasomal subunit Rpn1, but only Dsk2p's UBL has been shown to interact with the Rpn10 subunit of the proteasome (Ishii et al. 2006). These structural and binding differences between Dsk2p and Rad23 suggest that these two proteins may differ in the specificity of their recognition by and association with the proteasome. A crystal structure of the UBL domain of Dsk2p (Fig. 1) has been solved by X-ray diffraction method (Lowe et al. 2006). However, very little is known with regard to Dsk2p's interactions with various proteasomal components and possi...