The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

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)P2-rich PM critically requires its C2C and C2E domains and that the EC50 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+).