ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling

International audienceThe molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE. The plasma membrane of all eukaryotic cells undergoes constant renewal through repeated cycles of endocyto-sis and exocytosis. During endocytosis, cell surface proteins and lipids are internalized forming vesicular carriers which then merge with early endosomes, a process central to the regulation of nutrient uptake, cell surface receptor signaling, plasma membrane remodeling, cellular mobility and synaptic vesicle recycling 1. Most of these processes rely on clathrin-mediated endocytosis (CME) based on the clathrin machinery for shaping endocytic vesicles. However alternative pathways, collectively referred to as clathrin-independent endocytosis (CIE), also occur at the plasma membrane, although the molecular mechanisms leading to membrane bending and fission, as well as the biological significance of these pathways have yet to be clarified 2. An important advance in defining the molecular players involved in CIE came from two recent reports demonstrating that endophilin-A controls CIE of activated receptors including those for epidermal growth factor (EGF) and interleukin-2 (IL2), and that this pathway is hijacked by shiga-or cholera bacterial toxins 3,4. The three endophilin-A isoforms (A1, A2, A3) contain a Src Homology 3 (SH3) domain binding to both dynamin and synaptojanin 5 and a N-BAR (Bin/amphiphysin/Rvs) domain capable of sensing and generating membrane curvature 6. At synapses, Endophilins-A are known to be involved in both CME 7-9 and CIE 10. We have shown previously that besides dynamin and synaptojanin, a major interacting partner of endophilin-A is Alix (ALG-2-interacting protein X), first identified through its calcium-dependent binding to the penta-EF-hand protein ALG-2 (apoptosis-linked gene 2) 11,12. Alix is a 95 kD cytoplasmic protein with multiple interacting partners. The N-terminal Bro1-like domain 13 , binds the endosome-resident lipid, lysobisphosphatidic acid (LBPA) 14,15 and the charged multivesicular body protein 4B (CHMP4B) component of the endosomal sorting complex III required for transport (ESCRT-III) 16 , while the C-terminal long proline-rich domain (PRD) binds endophilin-A 12 , and also contains distinct interaction sites for ALG-2, the tumor suppressor gene 101 (Tsg-101) component of ESCRT-I, and Cbl Interacting protein-85 (CIN85) 17. The central region of Alix consists of a V-shaped domain composed of two triple-helical bundles which mediate protein dimerization 18. Alix i