Cytotoxic granule endocytosis depends on the Flower protein

Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. We studied the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis in Drosophila melanogaster. Flower is predominantly localized on intracellular vesicles that move to the synapse on target cell contact. Endocytosis is entirely blocked at an early stage in Flower-deficient CTLs and is rescued to wild-type level by reintroducing Flower or by raising extracellular calcium. A Flower mutant lacking binding sites for the endocytic adaptor AP-2 proteins fails to rescue endocytosis, indicating that Flower interacts with proteins of the endocytic machinery to mediate granule endocytosis. Thus, our data identify Flower as a key protein mediating granule endocytosis.