Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

► Rabex-59-73, harboring A20_ZF and MIU, binds linear, K63-linked and K48-linked Ub4 in vitro. ► Rabex-59-73 exhibits modest selectivity towards linear and K63-linked Ub4 over K48-linked one. ► MIU is critical in linkage-specific polyubiquitin recognition while A20_ZF is not. ► A20_ZF shows linkage-specific polyubiquitin selectivity distinct from orthologous ZnF4 of A20. ► A20_ZF and MIU may have differential roles in polyubiquitin recognition by Rabex-5 in vivo.