Kif5b-driven anterograde transport and clathrin-mediated endocytosis (CME) are responsible for opposite intracellular trafficking, contributing to plasma membrane homeostasis. However, whether and how the two trafficking processes coordinate remain unclear. Here, we show that Kif5b directly interacts with clathrin heavy chain (CHC) at a region close to that for uncoating catalyst (Hsc70) and preferentially localizes on large clathrin-coated vesicles (CCVs). Uncoating in vitro is decreased for CCVs from the cortex of kif5b conditional knockout (mutant) mouse and facilitated by adding CHC-binding Kif5b fragments, while cell peripheral distribution of CHC or Hsc70 keeps unaffected by Kif5b depletion. Furthermore, cellular entry of vesicular stomatitis virus that internalized into large CCV is inhibited in cells by Kif5b depletion or introducing a dominant-negative Kif5b fragment. These findings showed a new role of Kif5b in CCV uncoating and CME, indicating Kif5b as a molecular knot connecting anterograde transport to CME.

Role of anterograde motor Kif5b in clathrin-coated vesicle uncoating and clathrin-mediated endocytosis