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

• Published in: Cell chemical biology Vol. 24; no. 4; pp. 443 - 457.e6
• Main Authors: Chojnacki, Michal, Mansour, Wissam, Hameed, Dharjath S., Singh, Rajesh K., El Oualid, Farid, Rosenzweig, Rina, Nakasone, Mark A., Yu, Zanlin, Glaser, Fabian, Kay, Lewis E., Fushman, David, Ovaa, Huib, Glickman, Michael H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 20.04.2017
• Subjects: Binding Sites; Cross-Linking Reagents - chemistry; crosslinking; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Polyubiquitin - chemistry; Polyubiquitin - metabolism; proteasome; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Protein Binding; Protein Structure, Tertiary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; protein-protein interactions; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; ubiquitin; Ubiquitin - chemistry; Ubiquitin - genetics; Ubiquitin - metabolism; Ubiquitination - radiation effects; Ultraviolet Rays; proteasome; crosslinking; ubiquitin; protein-protein interactions
• ISSN: 2451-9456; 2451-9448; 2451-9456
• DOI: 10.1016/j.chembiol.2017.02.013

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The 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. [Display omitted] •Photoleucine was successfully incorporated into fully synthetic ubiquitin monomers•Embedded photoleucine permitted binding to the hydrophobic patch of ubiquitin•Enzymatically polymerized ubiquitin phototrap captured Ub-binding receptors•The first PC region of Rpn1, either isolated or proteasome-incorporated, bound polyUb Application of polyubiquitin-phototrap (polyUbPT), a novel set of chain-specific inducible photo-crosslinking probes, enables trapping of transient partners through the hydrophobic patch of ubiquitin. PolyUbPT captured Rpn1 from intact proteasome complexes. Rpn1 joins Rpn10 and Rpn13 as a proteasome subunit with affinity for polyUb and Ub-like domains.