Together, Rpn10 and Dsk2 Can Serve as a Polyubiquitin Chain-Length Sensor

Abstract

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.

Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

“…Notable also is that the conserved surface patch is distal to this interaction surface. This could mean either that the function of the patch is unrelated to protein-protein interaction or that TbBILBO1 makes contacts via two separate interfaces as reported previously for regulating ubiquitin substrate release (25,26). Future work will test these hypotheses in depth.…”

Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System

“…The perturbed residues cluster in and around the hydrophobic patch of Rub1 (Fig. 6D), similar to Ub-Rpn10 interaction (66), suggesting that Rub1 does bind the UIM domain of Rpn10, albeit weakly. Furthermore, the analysis of the titration curves did not yield a consistent K d because of the small CSPs observed.…”

“…To measure the affinity, titration curves were analyzed yielding a K d of 185 Ϯ 103 M for the distal Rub1 and of 36 Ϯ 9 M for the proximal Ub. Note that the latter K d value is essentially the same as for monoUb binding to UIM-Rpn10 (66). Moreover, the 15 N T 1 values show a lesser increase for the distal Rub1 than for the proximal Ub (Table I), suggesting that the UIM primarily binds to the latter.…”

“…NMR signals are highly sensitive to the local electronic environment in a molecule (63,64) and therefore have been widely used to map the interacting surfaces (13,14,58,65,66) and assess the strength of binding (14, 58, 59, 66 -68). A perturbation in the local environment in a protein could result in a shift of the NMR signal from its original position (referred to as chemical shift perturbation (CSP)) or broadening (attenuation) of the NMR signal, or both.…”

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

“…Thus, the protease activity of Ddi1 could possibly provide an alternative to the proteasome as a means to attack ubiquitylated proteins. Dsk2 is distinguished by the existence of an extraproteasomal pool that is largely complexed to a free pool of Rpn10 (van Nocker et al 1996;Matiuhin et al 2008;Zhang et al 2009a). In this complex, the UBL domain of Dsk2 binds the UIM element of Rpn10, which is the ubiquitin-binding element of Rpn10 (Zhang et al 2009a).…”