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) that 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 be a prerequisite for 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. Instead, depletion of VAMP8 inhibits recycling of endocytic cargoes and as exemplified here by transferrin receptor, skews their trafficking toward lysosomal degradation. VAMP8 depletion therefore indirectly impairs CCV formation and inhibits CME by depleting endocytic cargo. Overall, our study provides new insights into the crosstalk between endocytosis and endocytic recycling of CME cargo and demonstrates the critical role for cargo recruitment in stabilizing nascent CCPs to regulate CME. Significance StatementWe demonstrate that VAMP8 depletion inhibits recycling of endocytic cargo and reroutes transferrin receptors toward lysosomal degradation, thereby impairing endocytic vesicle formation and inhibiting clathrin-mediated endocytosis. Thus, the work uncovers a critical link between endocytic recycling and endocytosis, highlighting the importance of recycling pathways in maintaining membrane dynamics. However, the recruitment of VAMP8 into nascent clathrin-coated pits by CALM is not required for CME. While SNARE recruitment is essential for formation of COPI and COPII vesicles, our findings suggest that this requirement is not generalizable to endocytic CCVs.