Linkage-specific polyubiquitination regulates many cellular processes. The N-terminal fragment of Rabex-5 (Rabex-5(9-73)) contains tandem ubiquitin binding domains: A20_ZF and MIU. The A20_ZF-MIU of Rabex-5 is known to bind monoubiquitin but molecular details of polyubiquitin binding affinity and linkage selectivity by Rabex-5(9-73) remain elusive. Here we report that Rabex-5(9-73) binds linear, K63-and K48-linked tetraubiquitin (Ub(4)) chains with K-d of 0.1-1 mu M, determined by biolayer interferometry. Mutational analysis of qualitative and quantitative binding data reveals that MIU is more important than A20_ZF in linkage-specific polyubiquitin recognition. MIU prefers binding to linear and K63-linked Ub(4) with sub mu M affinities. However, A20_ZF recognizes the three linkage-specific Ub(4) with similar affinities with K-d of 3-4 mu M, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5. (C) 2012 Elsevier Inc. All rights reserved.