E-Syts, a family of membranous Ca2+-sensor proteins with multiple C2 domains

Abstract

C(2) domains are autonomously folded protein modules that generally act as Ca(2+)- and phospholipid-binding domains and/or as protein-protein interaction domains. We now report the primary structures and biochemical properties of a family of evolutionarily conserved mammalian proteins, referred to as E-Syts, for extended synaptotagmin-like proteins. E-Syts contain an N-terminal transmembrane region, a central juxtamembranous domain that is conserved from yeast to human, and five (E-Syt1) or three (E-Syt2 and E-Syt3) C-terminal C(2) domains. Only the first E-Syt C(2) domain, the C(2)A domain, includes the complete sequence motif that is required for Ca(2+) binding in C(2) domains. Recombinant protein fragments of E-Syt2 that include the first C(2) domain are capable of Ca(2+)-dependent phospholipid binding at micromolar concentrations of free Ca(2+), suggesting that E-Syts bind Ca(2+) through their first C(2) domain in a phospholipid complex. E-Syts are ubiquitously expressed, but enriched in brain. Expression of myc-tagged E-Syt proteins in transfected cells demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3. Structure/function studies showed that the plasma-membrane localization of E-Syt2 and E-Syt3 was directed by their C-terminal C(2)C domains. This result reveals an unexpected mechanism by which the C(2)C domains of E-Syt2 and E-Syt3 functions as a targeting motif that localizes these proteins into the plasma membrane independent of their transmembrane region. Viewed together, our findings suggest that E-Syts function as Ca(2+)-regulated intrinsic membrane proteins with multiple C(2) domains, expanding the repertoire of such proteins to a fourth class beyond synaptotagmins, ferlins, and MCTPs (multiple C(2) domain and transmembrane region proteins).

Fig. 1.

Domain structures of E-Syts. T, TMR; X, X domain unique to E-Syts; C₂A to C₂E, C₂ domains (note that the C₂C and C₂D domains of E-Syt1 are highly homologous to the C₂A and C₂B domains of all E-Syts). For an alignment of E-Syt sequences, see SI Fig. 5.

Fig. 2.

Ca²⁺- and phospholipid-dependent membrane binding by E-Syt2 C₂ domains. (A) Recombinant GST-fusion proteins containing the E-Syt2 domains indicated on the right were used in phospholipid-binding assays. Proteins were incubated in the absence of divalent cations, in 1 mM Ca²⁺, or in 1 mM Mg²⁺ with liposomes composed of 25% PS/75% PC (wt/wt). Liposomes were pelleted by centrifugation, and bound proteins were analyzed by SDS/PAGE and Coomassie staining. (B) Ca²⁺ dependence of phospholipid binding. Recombinant GST-fusion proteins containing either the E-Syt juxtamembranous X domain together with the C₂Aand C₂B domains (XC₂AB), or only the C₂A or C₂C domain from E-Syt2, were incubated with liposomes composed of 25% PS/75% PC in the presence of the indicated concentrations of free Ca²⁺. Proteins bound to the liposomes were analyzed by centrifugation, followed by SDS/PAGE and Coomassie blue staining. (C) Phospholipid dependence of Ca²⁺-dependent membrane binding of the E-Syt2 C₂AB domains. Phospholipid binding of a GST fusion protein containing the E-Syt2 C₂AB domains was tested at the indicated Ca²⁺ concentrations and in the presence of 1 mM Mg²⁺ (as a negative control) in liposomes with the four different phospholipid compositions shown on the right. (PIP, phosphatidylinositol phosphate; PE, phosphatidylethanolamine).

Fig. 3.

Subcellular localization of E-Syts in HEK293 cells. Confocal images of HEK293 cells transfected with various constructs expressing the proteins indicated on the left. (A) Synaptotagmin-7 (Syt-7). (B) Filamin-A. (C, E, and G) full-length E-Syt1, E-Syt2, or E-Syt3. (D, F, and H) truncated E-Syt1 (E-Syt1ΔTMR), E-Syt2 (E-Syt2ΔTMR), or E-Syt3 (E-Syt3ΔTMR) lacking the N-terminal TMR. All proteins contained an N-terminal myc-epitope. Transfected cells were fixed and stained with anti-myc antibodies (Left, red) and fluorescein-labeled phalloidin (Center, green). (Right) Merged images). Cells were analyzed either without detergent permeabilization to probe surface-exposed epitopes or after detergent solubilization with Triton X-100 as indicated on the right. (Scale bars: 5 μm.)

Fig. 4.

The C-terminal C₂ domain of E-Syt2 and E-Syt3 but not E-Syt1 direct plasma membrane localization. (A) Schematic diagram of the E-Syt2 deletion constructs and summary of their subcellular localization in transfected HEK293 cells (on right; PM, plasma membrane; Cyt, cytoplasmic). Numbers display residue numbers at the N and C termini of the various fragments. (B) Confocal images of HEK293 cells transfected with various constructs as indicated on the left, permeabilized, and stained with myc antibodies (red) and fluorescent phalloidin (green); merged images are depicted on the right (yellow indicates red/green overlap). (C) Confocal images of HEK293 cells transfected with myc-tagged C-terminal C₂ domains of E-Syt1 and E-Syt3, permeabilized, and stained with myc antibodies (red) and fluorescent phalloidin (green); merged images are depicted on the right (yellow indicates red/green overlap). (Scale bars for B and C, 5 μm.)