Abstract
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 Ca2+ and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca2+ sensor that penetrates membranes upon binding Ca2+ to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca2+ 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 (PIP2), but it is unclear whether PIP2 serves only as a membrane contact or enables specialized membrane-binding modes by these Ca2+ sensors. Furthermore, it has been shown that PIP2 uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca2+ influx, suggesting that current models for the action of these Ca2+ 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 Ca2+-dependent manner. Unexpectedly, we observed that PIP2 can drive membrane penetration by both syt1 and Doc2β in the absence of Ca2+, providing a plausible mechanism for Ca2+-independent, PIP2-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca2+, PIP2 drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca2+ sensor. Our results provide evidence of a novel role for PIP2 in regulating, and under some circumstances triggering, exocytosis.
Highlights
Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells
We observed that PIP2 can drive membrane penetration by both syt1 and Doc2 in the absence of Ca2؉, providing a plausible mechanism for Ca2؉-independent, PIP2-dependent exocytosis
A critical signal for exocytosis is Ca2ϩ [3], which acts upon tandem C2 domain proteins in the synaptotagmin (4 –8) and Doc2 [9, 10] families to trigger SNAREcatalyzed fusion of vesicular and plasma membranes [10, 11]
Summary
Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. A critical signal for exocytosis is Ca2ϩ [3], which acts upon tandem C2 domain proteins in the synaptotagmin (syt) (4 –8) and Doc2 [9, 10] families to trigger SNAREcatalyzed fusion of vesicular and plasma membranes [10, 11]. In the case of syt, binding to PIP2 under resting conditions “steers” the C2 domains of this protein, and its Ca2ϩ-dependent membrane-penetration activity, toward the plasma membrane to trigger release [13]. Our results define key biophysical differences between syt and Doc2 and provide a potential molecular mechanism by which PIP2 can directly trigger exocytosis in the absence of increases in [Ca2ϩ]i
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