Most mammalian proteins are secreted through the conventional secretory pathway, however a subset of cytosolic proteins are also secreted through unconventional protein secretion (UPS) pathways. We have previously described a novel player in the UPS of non-vesicular protein cargo, a widely expressed phospholipid scramblase called TMEM16F (1). Extracellular vesicles (EVs) embody a major pathway for UPS. Therefore, we are now investigating whether phospholipid scrambling is also important for vesicular UPS, and its impact on EV biogenesis, packaging and function.
To investigate a potential role for phospholipid scrambling in EV biology, we generated CRISPR/Cas9 TMEM16F knockouts in multiple mammalian cell lines. EV biogenesis/secretion were investigated using biochemical approaches and confocal microscopy and show that loss of TMEM16F reduces the number of EVs released per cell. Proteomic analysis shows that TMEM16F is present in EVs, and upon knockout, the protein content of EVs is altered. These finding were also confirmed by western blotting. Finally, we assessed the activity of EVs lacking TMEM16F and show that, in vitro the pro-proliferative properties of cancer-derived EVs are abolished.
Together these results suggest that phospholipid scrambling and membrane homeostasis is important for the UPS of both soluble and vesicular cargo. EV biogenesis/secretion is decreased when scrambling activity is abolished. Equally, the protein cargo carried by scramblase-deficient EVs is altered, and this leads to functional impairments. Together this data demonstrates phospholipid scramblase activity is an important regulator of EV biogenesis and function.