Unveiling physiological functions of extended synaptotagmins.

The endoplasmic reticulum (ER) is an extensive membrane network in eukaryotic cells important for many cellular processes. To orchestrate cellular functions, the ER forms junctions with other organelles, where the 2 membranes are closely tethered by protein-lipid and/or protein-protein interactions within 10–25 nm. Emerging evidence indicates that ER-organelle junctions are platforms for direct signal transduction and transfers of small molecules between the juxtaposed membrane compartments. At ER-plasma membrane (PM) junctions, proteins components, including the ER Ca2+ sensor STIM1, the PM Ca2+ channel Orai1, the lipid transfer protein Nir2, the vesicle-associated membrane proteins-associated proteins (VAPs), and the extended synaptotagmins (E-Syts), have been shown to participate in Ca2+ signaling and lipid metabolism (Fig. 1).1,2 Figure 1. Proteins Localized to ER-PM Junctions. E-Syt2 and E-Syt3 are constitutively localized to ER-PM junctions and mediate ER-PM tethering. E-Syt1, the Nir2-VAPs complex, and the STIM1-Orai1 complex are inducibly localized to ER-PM junctions following stimulation. ... E-Syts, E-Syt1/E-Syt2/E-Syt3, are ER proteins recently shown to mediate membrane tethering at ER-PM junctions.3,4 E-Syts contain an N-terminal ER membrane-associated region and a C-terminal cytosolic region consisting of a synaptotagmin-like-mitochondrial-lipid binding protein (SMP) domain and multiple C2 domains. The SMP domain forms a hydrophobic channel that binds glycerophospholipids and the C2 domains can mediate Ca2+-dependent binding to negatively-charged lipids. E-Syt2 and E-Syt3 contain 3 C2 domains and are constitutively localized to ER-PM junctions via C2 domain-dependent interactions with phosphatidylinositol 4,5-bisphosphate (PIP2) in the PM. Moreover, E-Syt2 is essential for the endocytosis of activated FGF receptor and its downstream signaling during Xenopus embryogenesis, and was shown to recruit the p21-GTPase activated kinase PAK1 to modulate cortical actin and cell adesion.5,6 On the other hand, E-Syt1 contains 5 C2 domains and is inducibly localized to ER-PM junctions following an elevation of cytosolic Ca2+.2,3 The translocation of E-Syt1 to ER-PM junctions facilitates the recruitment of Nir2 to ER-PM junctions and the replenishment of PM PIP2 following receptor-induced hydrolysis.2 Altogether, these findings suggest that the E-Syts not only mediate membrane tethering at ER-PM junctions but also contribute to cell signaling. Nevertheless, the physiological functions of the E-Syts remain unclear. In the recent paper by Herdman et al.,7 the authors generated E-Syt2/E-Syt3 double knockout mice to provide new insights into physiological functions of these proteins. They found that these mice are viable, developed normally and are fertile. This is in contrast to the essential role of E-Syt2 in mesoderm formation in early Xenopus embryos.6 With the structural similarity among the 3 E-Syts, it is plausible that widely expressed E-Syt1 may compensate the loss of E-Syt2 and E-Syt3 in mice. This possibility can be addressed by generating E-Syt1/E-Syt2/E-Syt3 triple knockout mice. It is also likely that the ER-PM tethering by E-Syt2 and E-Syt3 may be substituted by other proteins constitutively localized to ER-PM junctions in the double knockout animals. In yeast, ER-PM junctions are maintained by multiple families of tethering proteins that display functional redundancy.4 Significant loss of ER-PM junctions as well as disruption of cell signaling are found in “delta-tether” yeast cells lacking 6 different tethering proteins including the tricalbins (Tcb1/Tcb2/Tcb3, orthologs of E-Syts), Scs2/Scs22 (orthologs of VAPs), and Ist2 (an ER membrane protein related to the TMEM16 ion channel), but not in cells lacking only the tricalbins. Thus, it is of interest to examine the amount of ER-PM junctions in cells from the E-Syt2/E-Syt3 double knockout animals and investigate whether the ortholog of Ist2 and VAPs contribute to the tethering of ER-PM junctions in mammalian cells. Furthermore, new insights can be obtained by identifying additional proteins localized to ER-PM junctions. Interestingly, Herdman et al. showed that MEFs derived from E-Syt2/E-Syt3 double knockout mice are sensitive to stringent culture condition and to oxidative stress.7 Moreover, these MEFs exhibit clear defects in adhesion and migration. Notably, the “delta-tether” yeast cells are more sensitive to ER stress.4 It is possible that a decrease in ER-PM junctions in the MEFs derived from E-Syt2/E-Syt3 double knockout mice may lead to defective Ca2+ signaling and lipid metabolism, which are crucial for cell adhesion and migration. Alternatively, the endocytosis of cell surface receptors may be affected in these cells and animals. Future analyses of ER-PM junctions and cell signaling in E-Syt2/E-Syt3 double knockout mice will elucidate these possibilities.