Triggered Ca 2+ influx is required for extended synaptotagmin 1‐induced ER ‐plasma membrane tethering

The extended synaptotagmins (E‐Syts) are ER proteins that act as Ca 2+ ‐regulated tethers between the ER and the plasma membrane ( PM ) and have a putative role in lipid transport between the two membranes. Ca 2+ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi‐intact cells to buffers of variable Ca 2+ concentrations, we found that binding of E‐Syt1 to the PI (4,5)P 2 ‐rich PM critically requires its C2C and C2E domains and that the EC 50 of such binding is in the low micromolar Ca 2+ range. Accordingly, E‐Syt1 accumulation at ER ‐ PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca 2+ via its influx from the extracellular medium, such as store‐operated Ca 2+ entry in fibroblasts and membrane depolarization in β‐cells. We also show that in spite of their very different physiological functions, membrane tethering by E‐Syt1 ( ER to PM ) and by synaptotagmin (secretory vesicles to PM ) undergo a similar regulation by plasma membrane lipids and cytosolic Ca 2+ .