The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

• Published in: The EMBO journal Vol. 40; no. 6; pp. e106094 - n/a
• Main Authors: Pluska, Lukas, Jarosch, Ernst, Zauber, Henrik, Kniss, Andreas, Waltho, Anita, Bagola, Katrin, Delbrück, Maximilian, Löhr, Frank, Schulman, Brenda A, Selbach, Matthias, Dötsch, Volker, Sommer, Thomas
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 15.03.2021; John Wiley and Sons Inc
• Subjects: Assembly; Binding; cell stress; Chain branching; Computer Simulation; Conjugates; Deoxyribonucleic acid; DNA; DNA biosynthesis; Domains; Enzymes; Heat stress; Heat tolerance; Homology; K48‐linked; K63‐linked; Lysine; Models, Structural; NMR; Nuclear magnetic resonance; polyubiquitin; Polyubiquitin - biosynthesis; Protein Domains; Proteomics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Signal Transduction - physiology; Topology; Ubiquitin; Ubiquitin-Conjugating Enzymes - genetics; Ubiquitin-Conjugating Enzymes - metabolism; Ubiquitination; Ubiquitination - physiology; ubiquitin‐conjugating enzymes; Yeast; Yeasts; K63-linked; ubiquitin-conjugating enzymes; K48-linked; polyubiquitin; cell stress
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.2020106094

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis. SYNOPSIS Although ubiquitin‐conjugating enzymes Ubc1 and Ube2K have been studied in diverse biological contexts, the function of their UBA domain has remained poorly understood. Here, the UBA domain is found to facilitate the selective targeting of K63‐linked polyubiquitin chains, resulting in formation of branched K48/K63‐linked ubiquitin conjugates. UBA domain binding to an individual ubiquitin moiety in a K63‐linked chain enables reaction of the catalytic UBC domain with the adjacent proximal ubiquitin molecule. This process involves close association of the UBA and UBC domains, which are connected through a flexible linker region. The formation of K48/K63‐branched polyubiquitin is conserved from yeast to humans. Proteomics and genetic data indicate that branched chain formation by Ubc1 regulates resistance to heat stress and DNA replication stress. Binding of K63‐linked ubiquitin chains via a tightly‐linked accessory domains allows two homologous human and yeast E2 enzymes to add further K48‐linked moieties and build branched conjugates.