VAMP8 function reveals tight linkage between endocytic recycling and endocytosis

Clathrin-mediated endocytosis (CME) is a multistage process that involves the initiation and stabilization of clathrin-coated pits (CCPs), which invaginate and finally detach from the plasma membrane to form clathrin-coated vesicles (CCVs). Given that SNARE proteins are essential for downstream vesicle targeting and fusion events, their recruitment into nascent CCVs has been suggested to play a role in regulating CME progression. However, which and how SNARE proteins regulate CME remains to be explored. Here, we showed that siRNA-mediated knockdown of the R-SNARE, VAMP8 impairs CCP initiation, stabilization and invagination and strongly inhibits CME. Mechanistically, recruitment of VAMP8 to CCVs is not required for CME regulation. Instead, VAMP8 regulates CME indirectly by mediating endocytic recycling. Depletion of VAMP8 skews endosomal recycling of CME cargo, exemplified here by transferrin receptor, toward lysosomal degradation, thereby inhibiting CCV formation. Overall, our study provides new insights into the crosstalk between endocytosis and VAMP8-mediated endocytic recycling of CME cargo and demonstrates the significance of cargo recruitment in CME regulation.