1. Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141, Milan, Italy
2. Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, Connecticut 06510, USA
3. IFOM, The FIRC Institute for Molecular Oncology, 20134 Milan; and University of Milan, Medical School, 20122 Milan, Italy

Correspondence to: Pier Paolo Di Fiore^1,3 Correspondence and requests for materials should be addressed to P.P.D.F. (e-mail: Email: pdifiore@ieo.it).

Ubiquitination is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively^{1, 2, 3, 4}. Polyubiquitination targets proteins for destruction by the proteasome. The role of monoubiquitination is less understood, although a function in membrane trafficking is emerging, at least in yeast^{1, 3, 5}. Here we report that a short amino-acid stretch at the carboxy-termini of the monoubiquitinated endocytic proteins Eps15 and eps15R is indispensable for their monoubiquitination. A similar sequence, also required for this modification, is found in other cytosolic endocytic proteins, such as epsins and Hrs. These sequences comprise a protein motif, UIM (ref. 6), which has been proposed to bind to ubiquitin. We confirm this for the UIMs of eps15, eps15R, epsins and Hrs. Thus, the same motif in several endocytic proteins is responsible for ubiquitin recognition and monoubiquitination. Our results predict the existence of a UIM:ubiquitin-based intracellular network. Eps15/eps15R, epsins and Hrs may function as adaptors between ubiquitinated membrane cargo and either the clathrin coat or other endocytic scaffolds. In addition, through their own ubiquitination, they may further contribute to the amplification of this network in the endocytic pathway.