.............................................................. A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins Simona Polo*, Sara Sigismund*, Mario Faretta*, Monica Guidi*, Maria Rosaria Capua*, Giovanna Bossi*, Hong Chen†, Pietro De Camilli† & Pier Paolo Di Fiore*‡ * Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141, Milan, Italy † Department of Cell Biology and Howard Hughes Medical Institute, Yale UniversitySchool of Medicine, 295 Congress Avenue, New Haven, Connecticut 06510, USA ‡ IFOM, The FIRC Institute for Molecular Oncology, 20134 Milan; and University of Milan, Medical School, 20122 Milan, Italy ............................................................................................................................................................................. Ubiquitination is a post-translation modification in which ubi- quitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively 1–4 . Polyubiquitination targets proteins for destruc- tion 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 monoubiquitin- ated 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 ubiqui- tin. 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 func- tion 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. Eps15 is ubiquitinated after the activation of the epidermal growth factor (EGF) receptor (Fig. 1a, and ref. 7). This event is associated with an increase in the apparent relative molecular mass, M r , of ,8K, compatible with the molecular mass of a single ubiquitin molecule (Fig. 1a, right, and ref. 7). The related protein eps15R, for which several isoforms have been described 8 , is also ubiquitinated following EGF receptor activation (Fig. 1a). The M r of ubiquitinated eps15R is compatible with monoubiquitination of the isoforms migrating in the M r ,125 and ,108K region, whereas the 76K form is not modified (Fig. 1a). Eps15 and eps15R share the same modular architecture 9 com- prising (from amino to carboxy terminus) three EH domains, a coiled-coil region and a C-terminal region, which binds to the clathrin adaptor complex AP2 (Fig. 1b). We mapped the regions that are important for ubiquitination by employing an ubiquitina- tion assay and various fragments of the proteins as substrates. The C-terminal region, glutathione S-transferase (GST)–Cter, of both proteins contained all the determinants necessary for ubiquitina- tion (Fig. 1b). Eps15 and eps15R share little similarity in their C- terminal regions, with the notable exception of a stretch of about 40 amino acids at their ends 10 . We engineered deletions of the last 23 amino acids of eps15, either in the context of the holoprotein or of its C-terminal region, and tested their ubiquitination in vitro. We could not detect ubiquitination of the truncated proteins (Fig. 1c). Next, a series of mutants in the C-terminal region of eps15 were Figure 1 A motif in eps15 and eps15R responsible for monoubiquitination. a, B82L cells were either treated with EGF ( þ) or mock-treated ( 2). Total cellular lysates (4 mg) were immunoprecipitated (IP) and then immunoblotted (IB) with the indicated antibodies. The positions of eps15, monoubiquitinated eps15, and of the various isoforms of eps15R are indicated on the left. b, Top, scheme of the modular structure of eps15 and eps15R: EH domains, coiled-coil region and C-terminal region. Bottom, GST fusion proteins (0.3 mg) encompassing full length or fragments of eps15 (left panels) or eps15R (right panels) were subjected to an in vitro ubiquitination assay, using biotinylated ubiquitin (Methods). Detection was performed with horseradish-peroxidase-conjugated streptavidin or anti- GST, as indicated. c, GST fusion proteins, either full-length eps15 (FL) or fragments thereof (as indicated by the amino-acid positions above each lane) were subjected to the in vitro ubiquitination (Ub) assay, as in b. In b and c, relative molecular mass (M r ) markers are shown on the right. letters to nature NATURE | VOL 416 | 28 MARCH 2002 | www.nature.com 451 © 2002 Macmillan Magazines Ltd