This project is focused on understanding clathrin-independent forms of endocytosis. Endocytosis that occurs without clathrin coats occurs in all cells but is poorly understood. We are interested in studying the cargo proteins that enter cells by this mechanism, their intracellular itinerary once they have been internalized and whether they contain amino acid sequences that allow for specialized sorting within cells. We have been identifying new cargo proteins and found that a subset of these proteins take alternative traffic routes once they have entered cells. The major histocompatibility complex Class I protein (MHCI), is a prototypical clathrin-indepenent cargo protein and after internalization it reaches endosomes that contain cargo proteins such as the transferrin receptor that enter via clathrin-depenent endocytosis. From there, MHCI travels either to late endosomes and lysosomes where it is degraded or on to recycling tubules that return MHCI back to the cell surface. CD44, CD98, and CD147, however, show an altered itinerary in many cells where they traffic directly into the recycling tubules and avoid trafficking to late endosomal compartments. Consistent with this altered itinerary, CD44, CD98 and CD147 are long-lived proteins and are not degraded like MHCI, which is routed to late endosomes. We have now identified (Maldonado-Baez et al, 2013) sequences in the cytoplasmic domain of CD98 and CD147 that allow these cargo proteins to avoid trafficking to endosomes labeled with the early endosomal antigen 1 (EEA1) and lysosomes but instead traffic to recycling tubules. These sequences are transferrable and include di-acidic amino acid residues. We identified the microtubule and cargo tethering protein Hook1 as interacting with the cytoplasmic tails of CD98 and CD147 and responsible for this sorting function. In addition, we found that Hook1 works together with Rab22a and microtubules to sort CD98 and CD147 into recycling tubules. We are currently investigating other aspects of this sorting machine, such as identifying other components that link cargo sorting to tube formation and facilitation of recycling.