Proteasome subunit Rpn13 is a novel ubiquitin receptor

Abstract

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...

Protein Folding and Quality Control in the ER

“…), chain elongation factors (Ufd2, E4 ubiquitin ligase), and deubiquitinases (YOD1, Ataxin-3, VCIP135) (Liang et al 2006;Mueller et al 2008;Schuberth and Buchberger 2008;Ernst et al 2009). Ubiquitinated substrates are transferred to the proteasome by shuttle proteins, known as HR23A/B or Ubiquilin-1 (Rad23 and Dsk2 in yeast), which contain ubiquitinassociated (UBA) and ubiquitin-like (UBL) domains that bind to polyubiquitin chains and the proteasome subunits (Rpn10/13, Rpt5), respectively (Deveraux et al 1994;Lam et al 2002;Raasi and Wolf 2007;Husnjak et al 2008;Finley 2009;Lim et al 2009). …”

The E3 Ubiquitin Ligase Parkin Is Recruited to the 26 S Proteasome via the Proteasomal Ubiquitin Receptor Rpn13

“…One of the major aims of our research in recent years has been to identify novel Ub-and Ubl-binding proteins/domains and to determine their physiological role in the cell (35,43,44). We were specifically interested in parkin because the function of its N-terminal Ubl domain had not been completely characterized.…”

“…We have previously characterized the Pru domain at the N terminus of Rpn13 as a novel ubiquitin-binding domain, establishing Rpn13 as one of the major Ub receptors within the proteasome (Fig. 1A) (35,36). By using 1:1 interaction studies in yeast (Figs.…”

“…The proteasomal ubiquitin receptors Rpn10 (34) and Rpn13/ ADRM1 (35,36) can directly bind ubiquitinated cargo at the 19 S proteasome (see Fig. 1A).…”

hRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

“…The 19S portion is responsible for stimulating 20S to degrade proteins [9,10]. hRpn13 is a component of the 19S particle, serves as a receptor for both ubiquitin and the deubiquitinating enzyme UCH37, and can also enhance the activity of UCH37 [16,22]. Therefore, it can be inferred that hRpn13 has bi-directional roles in modulating UPP activity.…”