Granzyme B binds to target cells mostly by charge and must be added at the same time as perforin to trigger apoptosis

Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.