A, Domain architecture of AT3 protein. B, Multiple sequence alignments of the UIM motifs from AT3, S5a, Vps27 and RAP80. Note that the length of the linker region between the two tandem UIM motifs varies in these four proteins. The linker in AT3 is the shortest (6 residues), and then in RAP80 (∼11 residues), whereas those in S5a and Vps27p are longer with 50 and 23 residues, respectively. The sequences were aligned using ClustalW and represented using ESPript. C, Sequences of the linker regions for wild-type AT3-UIM12 and its mutants. GS-Sub, substitution with GSSGGS sequence; GS-Ins, Insertion of GSSGGS; RAP80-Linker, replacement with the linker sequence of RAP80.