Clathrin-independent endocytosis of ubiquitinated cargos The epidermal growth factor (EGF) receptor (EGFR) is internalized through clathrin-dependent and clathrin-independent pathways depending on EGF concentration. At low EGF concentrations, EGFR is internalized almost exclusively through the clathrin pathway and is not ubiquitinated. At higher EGF concentrations, a significant fraction of the receptor is internalized through a clathrin-independent, lipid-raft-dependent route, as the receptor becomes ubiquitinated. An EGFR mutant that cannot be ubiquitinated is internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, which signals through its ubiquitin moiety, is internalized exclusively through the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif (UIM), as shown by the ablation of three UIM-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization. Ubiquitination is a posttranslational modification that targets proteins to proteasomal degradation. Monoubiquitination functions as a signaling device through interactions with intracellular proteins harboring Ub-binding domains, such as the Ub-interacting motif (UIM). In mammals, the connection between monoubiquitination and internalization has remained elusive. The study focuses on the mechanisms of internalization of receptor tyrosine kinases, particularly the EGFR. The EGFR is monoubiquitinated at multiple sites through the action of the E3 enzyme Cbl. Although there is consensus on the function of Cbl and receptor ubiquitination in intracellular sorting of the EGFR, their role in the internalization step of endocytosis is less clear. To gain insight into this issue, a chimera was generated in which the extracellular and transmembrane domains of the EGFR are fused to a mutant Ub (Ubmut), unable to form polyUb chains. With this chimera, it was shown that ubiquitination is sufficient for internalization. The study elucidates the molecular mechanisms through which receptor ubiquitination directs internalization. The results show that a chimera encompassing the extracellular and transmembrane domain of the EGFR fused to Ub (EGFR/Ubmut) is constitutively internalized. By exploiting this chimera, the study determined how ubiquitination directs internalization. In EGFR-negative cells, the internalized EGFR/Ubmut colocalized extensively with caveolin-1, a marker of the non-clathrin lipid raft/caveolar pathway, but poorly with clathrin. By immunoelectron microscopy, EGFR/Ubmut was detected exclusively inClathrin-independent endocytosis of ubiquitinated cargos The epidermal growth factor (EGF) receptor (EGFR) is internalized through clathrin-dependent and clathrin-independent pathways depending on EGF concentration. At low EGF concentrations, EGFR is internalized almost exclusively through the clathrin pathway and is not ubiquitinated. At higher EGF concentrations, a significant fraction of the receptor is internalized through a clathrin-independent, lipid-raft-dependent route, as the receptor becomes ubiquitinated. An EGFR mutant that cannot be ubiquitinated is internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, which signals through its ubiquitin moiety, is internalized exclusively through the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif (UIM), as shown by the ablation of three UIM-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization. Ubiquitination is a posttranslational modification that targets proteins to proteasomal degradation. Monoubiquitination functions as a signaling device through interactions with intracellular proteins harboring Ub-binding domains, such as the Ub-interacting motif (UIM). In mammals, the connection between monoubiquitination and internalization has remained elusive. The study focuses on the mechanisms of internalization of receptor tyrosine kinases, particularly the EGFR. The EGFR is monoubiquitinated at multiple sites through the action of the E3 enzyme Cbl. Although there is consensus on the function of Cbl and receptor ubiquitination in intracellular sorting of the EGFR, their role in the internalization step of endocytosis is less clear. To gain insight into this issue, a chimera was generated in which the extracellular and transmembrane domains of the EGFR are fused to a mutant Ub (Ubmut), unable to form polyUb chains. With this chimera, it was shown that ubiquitination is sufficient for internalization. The study elucidates the molecular mechanisms through which receptor ubiquitination directs internalization. The results show that a chimera encompassing the extracellular and transmembrane domain of the EGFR fused to Ub (EGFR/Ubmut) is constitutively internalized. By exploiting this chimera, the study determined how ubiquitination directs internalization. In EGFR-negative cells, the internalized EGFR/Ubmut colocalized extensively with caveolin-1, a marker of the non-clathrin lipid raft/caveolar pathway, but poorly with clathrin. By immunoelectron microscopy, EGFR/Ubmut was detected exclusively in