Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.
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.
Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel; Department of Medical Biochemistry, Medical University of Łódź, Łódź, Poland.
Michal
M
Chojnacki
Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Wissam
W
Mansour
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands; Department of Chemical Immunology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
Dharjath S
DS
Hameed
Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA.
Rajesh K
RK
Singh
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
Farid
F
El Oualid
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
Rina
R
Rosenzweig
Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Mark A
MA
Nakasone
Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Zanlin
Z
Yu
The Technion Bioinformatics Knowledge Unit (BKU) of the Lorry Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Fabian
F
Glaser
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada.
Lewis E
LE
Kay
Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA. Electronic address: fushman@umd.edu.
David
D
Fushman
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands; Department of Chemical Immunology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Electronic address: h.ovaa@lumc.nl.
Huib
H
Ovaa
Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel. Electronic address: glickman@tx.technion.ac.il.
Michael H
MH
Glickman
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D062105:Molecular Docking Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000528:radiation effects; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex
crosslinking; proteasome; protein-protein interactions; ubiquitin