Syntenin and CD63 Promote Exosome Biogenesis from the Plasma Membrane by Blocking Cargo Endocytosis.

Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the endosomal biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosome-localized CD63. We find instead that syntenin blocks CD63 endocytosis, allowing CD63 to accumulate at the plasma membrane, thereby increasing its direct budding from the plasma membrane. Consistent with this model, we show that specific and general inhibitors of endocytosis induce the exosomal secretion of CD63 and that endocytosis signals inhibit the vesicular secretion of exosome cargo proteins. Furthermore, we show that CD63 is itself a competitive inhibitor of AP-2-mediated endocytosis, driving the plasma membrane accumulation and exosomal secretion of itself and other lysosome membrane proteins. Our results support the hypothesis that highly-enriched exosome cargo proteins bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, and that syntenin and CD63 regulate the loading of lysosomal proteins into exosomes by an Alix-independent modulation of endocytosis.