Phosphatidylinositol 4,5-bisphosphate drives Ca-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2β.

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca sensor that penetrates membranes upon binding Ca to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2β bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP), but it is unclear whether PIP serves only as a membrane contact or enables specialized membrane-binding modes by these Ca sensors. Furthermore, it has been shown that PIP uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca influx, suggesting that current models for the action of these Ca sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2β, like syt1, penetrates membranes in a Ca-dependent manner. Unexpectedly, we observed that PIP can drive membrane penetration by both syt1 and Doc2β in the absence of Ca, providing a plausible mechanism for Ca-independent, PIP-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca, PIP drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca sensor. Our results provide evidence of a novel role for PIP in regulating, and under some circumstances triggering, exocytosis.