The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIMdependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.The UIM 1 was first described as a peptide sequence consisting of a highly conserved ⌽-X-X-A-X-X-X-S-X-X-Ac core where ⌽ represents a hydrophobic residue and Ac is an acidic residue (1). It was identified based on the ubiquitin binding region of the RPN10 subunit of the 26 S proteasome (2, 3). The presence of UIMs in numerous proteins ranging from the MachadoJoseph disease protein (MJD1/ataxin3) to USP25, a member of the deubiquitylating enzyme family, suggests that this region is involved in regulating protein function. Indeed our previous studies and those of others have demonstrated an important role for UIMs in both ubiquitylation and in ubiquitin binding (2-11).Ubiquitylation is a post-translational modification resulting in the covalent attachment of ubiquitin through its COOHterminal Gly to the ⑀-NH 2 group of a Lys residue in a target protein. This process involves a multienzyme cascade that begins with the activation of ubiquitin in the presence of ATP and an E1 ubiquitin-activating enzyme. Subsequently the ubiquitin is transferred through a thiol-ester bond to a ubiquitin-conjugating enzyme (E2) and through the action of an E3 ubiquitin ligase is attached to the substrate by an isopeptide bond. Polyubiquitylation, the attachment of multimeric chains of ubiquitin, leads to the proteolytic destruction of proteins when Lys 48 of ubiquitin is the site of chain formation. However, ubiquitin chains formed through Lys 63 are not involved in protein degradation but rather a variety of processes including DNA repair, translation, IB kinase activation, endocytosis, and protein transport (for a review, see Ref. 12). In contrast to po...

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