CD63 regulates cholesterol storage within endosomes and its distribution via exosomes

Abstract Exosomes are small extracellular vesicles that are enriched in specific proteins and lipids during their generation as intraluminal vesicles in multivesicular endosomes. Contrary to proteins, the endosomal sorting mechanisms of lipids on intraluminal vesicles are still ill-defined. Here, we find that the tetraspanin CD63, highly enriched in ILVs and exosomes, regulates cholesterol sorting on ILVs and exosomes. This process generates a pool of cholesterol that affects exosomes physical properties and can be mobilized by the NPC1/2 complex from ILVs and exosomes. Absence of CD63 redirects cholesterol from endosomes to the Trans-Golgi Network by actin dependent vesicular transport, placing CD63 and cholesterol at the center of a balance between inward and outward budding of endomembranes. These results establish CD63 as cholesterol sorting mechanism within endosomes and place ILVs and exosomes as alternative providers of cholesterol.