Protein ubiquitination can have markedly different consequences depending on whether substrates are modified with a single ubiquitin (monoubiquitination) or a ubiquitin chain that contains several ubiquitin molecules (polyubiquitination). Polyubiquitination generally targets proteins for proteasome-dependent degradation, whereas monoubiquitination serves nonproteolytic functions in diverse cellular processes such as endocytosis, protein sorting within the secretory pathway, chromatin remodeling and viral budding. Surprisingly, ubiquitin pathway enzymes that promote efficient substrate polyubiquitination are also required for monoubiquitination of certain substrates. So how does the cell discriminate between targets destined for polyubiquitination and those for monoubiquitination?Some insight into this conundrum comes from the data of Di Fiore and colleagues, who demonstrate that a protein motif called the ubiquitin interaction motif (UIM) is necessary for monoubiquitination [1xA single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Polo, S. et al. Nature. 2002; 416: 451–455Crossref | PubMed | Scopus (478)See all References][1].The authors embarked on defining the sequence elements necessary for monoubiquitination of two proteins of the endocytic pathway, eps15 and eps15R. Using in vitro and in vivo ubiquitination assays, they identified a conserved 40-amino-acid stretch at the extreme C-termini of eps15 and eps15R as indispensable for monoubiquitination. Subsequently, a database search revealed the C-terminal sequence in eps15 and eps15R to be highly related to the previously defined UIM motif. The authors further found that UIMs present in other endocytic proteins such as epsin and Hrs are also necessary for monoubiquitination. Since UIMs are thought to confer a ubiquitin binding property, the authors next tested the ubiquitin binding capacity of their UIM-containing proteins. Indeed, UIMs in eps15, epsin and Hrs were able to bind to both monoubiquitin and ubiquitinated proteins from cell extracts.Is the ubiquitin binding function of UIM linked to the role of UIM in monoubiquitination? To address this question, they tested the effects of mutating the UIMs in eps15 and found that mutation of one of the two UIMs in eps15 abolished both monoubiquitination and interaction with ubiquitin. These findings led the authors to suggest that the two properties of the UIM motif are indeed functionally coupled.How might UIMs negatively regulate ubiquitin chain length? The authors propose a model in which UIMs would initially contribute to interactions between the substrate and the ubiquitination apparatus. However, following ubiquitination, UIMs would contact the appended ubiquitin – in cis or in trans on other monoubiquitinated proteins – and block the addition of further ubiquitin moieties. UIMs are present in a wide spectrum of proteins, including ubiquitin pathway enzymes. It remains to be seen whether this protein motif represents a general mechanism for monoubiquitination that can be extended to non-endocytic substrates, such as histones, or whether it has evolved as a specific regulator of ubiquitination in the endocytic pathway.