Interaction Landscape of K29-Linked Ubiquitin Signaling Revealed by a Linkage-Specific Synthetic Antigen-Binding Fragment

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells (Tsuchiya et al., 2018). However, the lack of specific binders as tools to detect K29-linked polyubiquitin has prevented further understanding of its function. In this study, we screened and characterized a synthetic antigen-binding fragment that can specifically recognize K29-linked polyubiquitin. We determined the crystal structure of this fragment bound to K29-linked diubiquitin, which revealed the molecular basis of sAB-K29 specificity. We identified the involvement of K29-linked ubiquitination in RNA processing, the proteotoxic stress response and cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1 phase.