Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin via a conserved N-terminal region termed the Pru domain (Pleckstrin-like receptor for ubiquitin), which binds K48-linked diubiquitin with an affinity of ∼90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like domains of the UBL/UBA family of ubiquitin receptors. A synthetic phenotype results in yeast when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Since Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome.In eukaryotes, selective protein degradation is performed primarily by the ubiquitinproteasome pathway. The 26S proteasome is a huge macromolecular machine that contains a proteolytically active 20S core particle (CP) capped at one or both ends by a 19S regulatory particle (RP)1. The RP recognizes ubiquitinated substrates, deconjugates ubiquitin chains, and unfolds substrates prior to their translocation into the CP. Proteasome subunit Rpn10/ S5a was shown to bind ubiquitin chains via ubiquitin-interaction motifs (UIMs)2. Receptors were subsequently identified that are not integral proteasome subunits, but deliver ubiquitinated targets to the proteasome (for reviews, see 5 and 6). Canonical members of this UBL/UBA family of receptors are Rad23 (hHR23a/b in humans), Dsk2 (hPLIC-1/2 in humans) and Ddi13 , 4 , 7 -9. UBA domains bind ubiquitin [10][11][12] and UBL domains interact reversibly with the proteasome, principally via Rpn1, but potentially also via Rpn10 13-15 . Using a yeast two-hybrid screen, with a bait of ubiquitin lacking the last two glycines to prevent its conjugation 25 , we identified the N-terminal segment of human Rpn13 (hRpn13) as a ubiquitin-binding partner. The interaction was confirmed using murine Rpn13 (mRpn13) as bait against monoubiquitin and Rpn2 as prey ( Figure 1A). Rpn13 from S. cerevisiae (scRpn13) aligns with the ubiquitin-binding N-terminal region of hRpn13 ( Figure 1B). Comprehensive sequence analysis using profiles and Hidden Markov Models failed to reveal similarity to known ubiquitin-or proteasome-binding motifs ( Figure 1C and data not shown). Deletion mutants encompassing residues 1-150 were tested for tetraubiquitin binding, thus mapping the minimal binding domain to residues 1-130 ( Figure 1D). Although smaller fragments of mRpn13 also showed detectable binding to ubiquitin, they were unstable and expressed poorly as GST-fusions.The significance of the ubiquitin-Rpn13 interaction would be supported if it were conserved from yeast to mammals, particularly as budding yeast Rpn13 is truncated and the conserved N-terminal region ( Figure 1C) only 25% id...

Targeted protein degradation is largely performed by the ubiquitin-proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a 1 , which is not essential for ubiquitin-mediated protein degradation in budding yeast 2 . In the accompanying manuscript we report that Rpn13 3-7 , a component of the 9-subunit proteasome base, functions as a ubiquitin receptor 8 , complementing its known role in docking deubiquitinating enzyme Uch37/UCHL5 [4][5][6] to the proteasome. Here, we merge crystallography and NMR data to describe Rpn13's ubiquitin binding mechanism. We determined the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for Rpn13's role as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13's role as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37. The structure of murine Rpn13 (mRpn13) (1-150) was determined at 1.7 Å resolution by Xray crystallography, and found to contain a Pleckstrin Homology (PH) domain fold ( Figure 1a and 1b) (Structure determination and refinement statistics are provided in the Supplement). In particular, whereas the first 21 N-and last 20 C-terminal amino acids are unstructured, residues 22-130 form a PH domain fold. This result was surprising, as primary sequence alignment did not identify Rpn13 to be homologous to previously characterized proteins. This finding coupled with its ubiquitin receptor properties 8 prompted us to name the N-terminal domain of Rpn13 Pleckstrin-like receptor for ubiquitin (Pru).Though very divergent at their sequence level, all PH domains have a common β-sandwich fold. The PH domain of Rpn13 is composed of a 4-stranded twisted antiparallel β-sheet (β 1-4 : residues 22-34, 45-52, 56-62, 71-74) that packs almost orthogonally against a second triple stranded β-sheet (β 5-7 : residues 80-85, 92-98, 103-110) (Supplementary Figure 1). Similar to other PH domains, Rpn13 Pru forms a hydrophobic core containing conserved hydrophobic residues (F26, V47, I49, F59, F82, Y94, L96, F107 and M109), which are located within β-sheets. One end of the β-sandwich is capped by a long Cterminal amphipathic α-helix (residues 117-128), which is stabilized by interactions between V124 and L128, whereas the other corner of the hydrophobic core is closed by three loops formed by residues located between strands S1/S2, S3/S4 and S6/S7 ( Figure 1a and Supplementary Figure 1).Despite much effort, we were unable to crystallize the Rpn13 Pru:ubiquitin complex; however, we were able to...

Summary Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin interacting motifs (UIMs). Here, we use NMR and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the “proximal” one) is covalently bonded to G76 of the other (the “distal” subunit). We demonstrate that S5a’s UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is highly adaptive and cooperative towards binding ubiquitin chains.

Proteasome–ubiquitin receptor hRpn13/Adrm1 binds and activates deubiquitinating enzyme Uch37/UCHL5 and is targeted by bis-benzylidine piperidone RA190, which restricts cancer growth in mice xenografts. Here, we solve the structure of hRpn13 with a segment of hRpn2 that serves as its proteasome docking site; a proline-rich C-terminal hRpn2 extension stretches across a narrow canyon of the ubiquitin-binding hRpn13 Pru domain blocking an RA190-binding surface. Biophysical analyses in combination with cell-based assays indicate that hRpn13 binds preferentially to hRpn2 and proteasomes over RA190. hRpn13 also exists outside of proteasomes where it may be RA190 sensitive. RA190 does not affect hRpn13 interaction with Uch37, but rather directly binds and inactivates Uch37. hRpn13 deletion from HCT116 cells abrogates RA190-induced accumulation of substrates at proteasomes. We propose that RA190 targets hRpn13 and Uch37 through parallel mechanisms and at proteasomes, RA190-inactivated Uch37 cannot disassemble hRpn13-bound ubiquitin chains.