Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Abstract

SUMMARY Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and the polyUb chains to a range of cellular protein targets. Best documented Ub signaling pathway is the one that delivers polyUb-proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb-conjugates. Our work demonstrates the value of UbPT and we expect that its future uses will help define and investigate the ubiquitin interactome.

NEDDylation promotes nuclear protein aggregation and protects the Ubiquitin Proteasome System upon proteotoxic stress

“…One microgram of purified 26S proteasomes was used in an in vitro processing/degradation assay with 300 ng of purified His 6 -tagged conjugates in 40 μl reactions containing 25 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 10% glycerol, 1 mM ATP, 1 mM DTT at 37 °C as described in ref. 66 . Reactions were stopped by the addition of 40 μl of 2× SDS Laemmli’s buffer and analysed by western blotting.…”

Resolving the Complexity of Ubiquitin Networks

“…Once photoactivated by UV, it is able to crosslink nearby proteins, thus stabilizing often very weak UBD:Ub interactions. By using Ub-PT as a tool, Chojnacki et al (2017) identified proteasomal subunit Rpn1 as a novel Ub receptor. Moreover, Ub-PT can have pLeu incorporated at multiple positions within Ub molecule, making it very useful for capturing UBDs that interact in different ways with Ub.…”

Development and application of ubiquitin-based chemical probes

“…Ovaa et al introduced photoleucine containing a photocrosslinking group into the sequence of Ub through SPPS, and then enzymatically assembled it into a Ub chain to generate a photocrosslinking polyUb probe. 97 3.4.2.2 Insertion of unnatural amino acids. Virdee et al used an evolved engineered pair of tRNA and aminoacyl-tRNA synthetase to embed p-benzoyl-L-phenylalanine (Bpa) into the sequence of Ub.…”

“…98 Meanwhile, Glickman et al developed photo-crosslinking polyUb probes for detecting proteasome subunits that interact with Ubs. 97 4.2.4 Monitoring changes of Ub chain conformation. Komander et al designed M1, K63 and K48 diUb probes containing a FRET dye pair.…”