Mixed-Linkage Ubiquitin Chains Send Mixed Messages

Research on ubiquitin (Ub) signaling has focused primarily on homogeneously linked polyUb. Although polyUb containing different linkages within the same chain exist, their structures and signaling properties are unknown. These mixed-linkage chains could be unbranched (i.e., no more than one lysine or methionine linkage per Ub) or branched. Here, we examined the structure, dynamics, receptor selectivity, and disassembly of branched and unbranched tri-Ub containing both K48 and K63 linkages. Each linkage was virtually indistinguishable from its counterpart in homogeneously linked polyUb. Linkage-selective receptors from hHR23A and Rap80 preferentially bound to the K48 or K63 linkages in the branched trimer. Linkage-selective deubiquitinases specifically cleaved their cognate Ub-Ub linkages in mixed-linkage chains, and the 26S proteasome recognized and processed branched tri-Ub. We conclude that mixed-linkage chains retain the distinctive signaling properties of their K48 and K63 components and that these multiple signals can be recognized by multiple linkage-specific receptors. Finally, we propose a new, comprehensive notation for Ub and Ub-like polymers. [Display omitted] ► K48 and K63 linkages in branched and unbranched tri-Ub retain their structural features ► Linkage-selective receptors recognize K48 and K63 linkages in the branched tri-Ub ► Linkage-specific deubiquitinases cleave their cognate Ub-Ub linkages in the mixed-linkage chains ► The 26S proteasome recognizes and processes branched K48- and K63-linked tri-Ub The outcomes of linkage mixing in polyubiquitin are poorly understood. Nakasone et al. show that Lys48 and Lys63 linkages retain their distinctive structural and signaling properties in mixed-linkage branched and unbranched chains, suggesting a possibly of encoding multiple signals in the same polyubiquitin chain.