Endocytosis of membrane receptors: two pathways are better than one.

E ndocytosis pathways are used by eukaryotic cells for internalization of nutrients, signal transduction regulation, and modulation of plasma membrane composition. The cytoplasmic regions of plasma membrane ‘‘cargo’’ proteins harbor signals that are recognized by ‘‘cargo adaptor’’ proteins; this concentrates the cargo proteins at sites of budding for subsequent internalization via an endocytic vesicle. There are several endocytic pathways that can mediate the internalization of many different cargo proteins. The best-studied pathway is clathrin-dependent endocytosis (CDE), defined by a requirement for the protein clathrin, which is the major component of the endocytic vesicle coat. There are multiple clathrin-independent endocytosis (CIE) pathways that generally depend on cholesterol-rich membrane domains (i.e., rafts, which can also be encased by the protein caveolin to form membrane invaginations called caveolae). Cargo internalization signals are short peptide motifs; yeast also uses posttranslationally conjugated ubiquitin (Ub) moieties (1). In this issue of PNAS, two studies show that Ub can act as an endocytic signal in mammalian cells; in the absence of clathrin, Ub can direct entry via a CIE pathway (2, 3). The idea that ubiquitylated cargo can travel via a CIE pathway was previously unrecognized, probably because the endocytic factors that can recognize and bind Ub (epsin and Eps15) were first shown to play key roles in the CDE pathway. Now, it seems that these endocytic factors may play multiple roles, depending on which signal the cargo protein presents, or which endocytic route is being used. In recent years it has become clear that, in addition to its well known roles in proteasome-dependent protein degradation, Ub is also involved in protein trafficking and membrane protein internalization (4, 5). Ub not only modifies cargo proteins to act as an endocytic signal; components of the endocytic machinery are also reversibly ubiquitylated, possibly regulating their activities or interactions (6, 7). These trafficking roles for Ub require its Ile-44 residue, and are mediated by Ub-binding modules such as Ub interaction motifs (UIM) or Ub associated (UBA) domains, which are conserved features of the epsin and Eps15/R endocytic proteins (7–10). These proteins bind each other and to the cargo adaptor AP2; epsins and AP2 also directly bind clathrin. Ub-mediated mechanisms seem to be particularly important for endocytosis and regulation of growth factor receptors (11); many studies focus on the EGF receptor (EGFR), a tyrosine kinase receptor. In the absence of the EGF ligand, 60% of EGFR is located at caveolae and noncaveolae rafts. Upon