Ca 2+ releases E‐Syt1 autoinhibition to couple ER ‐plasma membrane tethering with lipid transport

Article8 December 2017free access Transparent process Ca2+ releases E-Syt1 autoinhibition to couple ER-plasma membrane tethering with lipid transport Xin Bian orcid.org/0000-0002-5501-0471 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Yasunori Saheki Corresponding Author [email protected] orcid.org/0000-0002-1229-6668 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Pietro De Camilli Corresponding Author [email protected] orcid.org/0000-0001-9045-0723 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Xin Bian orcid.org/0000-0002-5501-0471 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Yasunori Saheki Corresponding Author [email protected] orcid.org/0000-0002-1229-6668 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore Search for more papers by this author Pietro De Camilli Corresponding Author [email protected] orcid.org/0000-0001-9045-0723 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Author Information Xin Bian1,2,3,4, Yasunori Saheki *,1,2,3,4,5 and Pietro De Camilli *,1,2,3,4,6 1Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA 3Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA 4Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT, USA 5Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA *Corresponding author. Tel: +65 6592 3996; Fax: +65 6515 0417; E-mail: [email protected] *Corresponding author. Tel: +1 203 737 4461; Fax: +1 203 737 4436; E-mail: [email protected] EMBO J (2018)37:219-234https://doi.org/10.15252/embj.201797359 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis The C2A and C2C domains of extended synaptotagmin 1 (E-Syt1) regulate both its lipid transport and membrane tethering activity in a Ca2+-dependent manner. The C2A and C2C domain of E-Syt1 mediate different actions of Ca2+ on E-Syt1 function. The C2A domain interacts with the SMP domain to inhibit lipid transport. Ca2+ releases the C2A-SMP domain interaction. Binding of Ca2+ to the C2C domain allows efficient binding to the PI(4,5)P2-rich plasma membrane via the C2E domain. Both Ca2+ binding sites are required for the regulation of lipid homeostasis at the plasma membrane. Introduction Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites represent a general feature of all eukaryotic cells (Friedman & Voeltz, 2011; Gallo et al, 2016; Saheki & De Camilli, 2017a). Their occurrence reflects the presence of proteins that tether the two membranes and mediate cross talk between them. One such class of tethers is the extended synaptotagmins (E-Syts), resident proteins of the ER membrane that are evolutionarily conserved from unicellular organisms to all metazoans (Craxton, 2001, 2007; Manford et al, 2012; Toulmay & Prinz, 2012; Giordano et al, 2013; Levy et al, 2015; Perez-Sancho et al, 2015). In mammals, they are encoded by three different genes, E-Syt1, E-Syt2, and E-Syt3 (Min et al, 2007), which form homo- and heterodimers. All three E-Syts comprise an N-terminal hydrophobic hairpin through which they are anchored to the ER (Giordano et al, 2013; Saheki et al, 2016). This region is followed by a synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain (Lee & Hong, 2006; Kopec et al, 2010; Toulmay & Prinz, 2012; Schauder et al, 2014) and multiple C2 domains, five in E-Syt1 and three in E-Syt2 and E-Syt3 (see Fig 1A; Min et al, 2007; Saheki & De Camilli, 2017b). As shown by the crystal structure, the first two C2 domains of E-Syt2 (C2AB) are arranged in tandem (Schauder et al, 2014; Xu et al, 2014) and a similar arrangement is predicted for the C2AB domains of E-Syt1 and E-Syt3. The last C2 domain (C2E for E-Syt1 and C2C for E-Syt2 and E-Syt3) is characterized by a positively charged surface (Idevall-Hagren et al, 2015), and the additional C2 domains unique to E-Syt1 (C2CD) are similar to C2AB and most likely represent a duplication of this pair (Min et al, 2007). The recruitment of E-Syts to ER-PM contact sites requires PI(4,5)P2 in the PM and occurs constitutively in the case of E-Syt2 and E-Syt3, while it requires elevation of cytosolic Ca2+ in the case of E-Syt1 (Chang et al, 2013; Giordano et al, 2013; Fernandez-Busnadiego et al, 2015; Idevall-Hagren et al, 2015). Figure 1. E-Syt1cyto specifically binds to PI(4,5)P2-containing membranes in a C2E-dependent way in the absence of Ca2+ and C2C, but not C2A, stimulates this binding in the presence of Ca2+ A. Domain structures of E-Syts (left) and E-Syt1 constructs (right) used for the liposome tethering assays shown in the figure. Slanted white lines indicate the mutations in the Ca2+ binding sites in C2A and C2C domains. B–D. Liposome aggregation, due to tethering of anchoring and target liposomes in the presence of E-Syt1 constructs (protein:lipid ratio 1:1,000) at RT, as assessed by increase in turbidity (OD 405 nm). Time-courses are at left, and bar graphs showing quantification of OD405 increases at the end of the incubation (arrows in the left panel) are at right. (B) Effect of the lipid composition and C2E on liposome tethering by E-Syt1cyto in the absence of Ca2+. A cocktail of EGTA, imidazole, and proteinase K (Cocktail) was added after 10 min. (C) Effect of the absence or presence of Ca2+ on liposome tethering by E-Syt1cyto with or without mutations in the Ca2+-binding sites in C2A and C2C. (D) Effect of the absence or presence of Ca2+ and of PI(4,5)P2 in the target liposomes on liposome tethering by E-Syt1cyto lacking the C2E domain. Mean and SD of three independent experiments. P-values from t-test with Bonferroni corrections are quoted on the graphs. ****P < 0.0001; n.s., not significant. Download figure Download PowerPoint The defining feature of the E-Syts among proteins with multiple C2 domains is the presence of the SMP domain (Lee & Hong, 2006), a member of the TULIP domain superfamily (Kopec et al, 2010). A shared characteristic of TULIP domains, which are present both in extracellular and intracellular proteins, is the property to harbor lipids within a hydrophobic cavity and, at least in many cases, to transport them (Oram et al, 2003; Qiu et al, 2007; Kopec et al, 2011; Schauder et al, 2014; AhYoung et al, 2015; Alva & Lupas, 2016; Jeong et al, 2016; Saheki et al, 2016; Yu et al, 2016; Lees et al, 2017; Liu et al, 2017). SMP domains are typically found in intracellular proteins that act at membrane contact sites (Kornmann et al, 2009; Toulmay & Prinz, 2012; Reinisch & De Camilli, 2016; Lees et al, 2017; Liu et al, 2017). Structural and biochemical studies of the SMP domain of E-Syt2 revealed that it dimerizes to form a 90-Å-long cylinder with a deep hydrophobic groove that runs along its main axis and that contains glycerophospholipids (Schauder et al, 2014). Consistent with these properties, purified recombinant E-Syt1 transfers glycerolipids between two populations of liposomes that mimic the ER and the PM, respectively (Saheki et al, 2016; Yu et al, 2016), without selectivity for a specific headgroup (Schauder et al, 2014; Hoglinger et al, 2017). Genome-edited cells lacking all the three E-Syts showed no major differences in the steady-state glycerolipids compositions of the PM. However, delayed clearance of the transient accumulation of diacylglycerol (DAG) at the PM produced by phospholipase C (PLC)-dependent PI(4,5)P2 hydrolysis was observed (Saheki et al, 2016), suggesting a role of E-Syts in the homeostatic response that follows a stimulus. This phenotype was rescued by expression of E-Syt1, but not by E-Syt1 lacking the SMP domain. Additionally, rescue required an elevation in cytosolic Ca2+ (Saheki et al, 2016). Whether other changes at the PM occur in response to stimuli in the E-Syts KO cells remain unclear. Of the five C2 domains in E-Syt1, two (C2A and C2C) bind Ca2+ (Min et al, 2007). Ca2+ binding to the C2C domain is responsible for ER tethering to the PM, as mutations that impair its Ca2+ binding were sufficient to abolish Ca2+-dependent recruitment of E-Syt1 to the PM (Chang et al, 2013; Giordano et al, 2013). The mechanism involved in this effect is not fully understood yet. It may be mediated by (i) a direct interaction of the C2C domain with the PI(4,5)P2-rich PM (Giordano et al, 2013), (ii) the release by Ca2+ of an inhibitory action of the C2C domain on the binding of C2E to the PI(4,5)P2-rich PM (Idevall-Hagren et al, 2015), or (iii) both mechanisms. In addition, mutations in the Ca2+-binding sites of either the C2A or the C2C domain reduced the lipid transfer activity of E-Syt1 in vitro (Yu et al, 2016). However, the function of C2A in SMP domain-dependent lipid transfer remains elusive. Interestingly, the C2A domain of E-Syt2 and E-Syt3, two proteins whose binding to the PM is not regulated by Ca2+, also contains Ca2+-binding sites. A plausible scenario is that the C2A domain of the E-Syts may play a regulatory role in lipid transfer independent of tethering. Goal of this study was to test this hypothesis. Here, we find that the Ca2+-dependent properties of the C2A and C2C domains of E-Syt1 have different functions at membrane contact sites. Using a liposome-based assay, we confirm that Ca2+ binding to the C2C domain acts primarily by enabling the binding of the C2E domain to PI(4,5)P2-rich membranes. Importantly, we show that Ca2+ binding to the C2A domain enables lipid transfer by E-Syt1 via the release of an autoinhibitory intramolecular interaction of this domain with the SMP domain. We also show that Ca2+ binding to the C2A and C2C domains plays important roles in the E-Syt1-dependent regulation of lipid homeostasis at the PM in living cells. Results C2C and C2E domains of E-Syt1 cooperate in membrane tethering The contributions of individual C2 domains in E-Syt1-mediated membrane tethering were assessed using an in vitro liposome turbidity assay (Saheki et al, 2016). To this aim, purified cytosolic fragment of human E-Syt1, in which the N-terminal region (including the hydrophobic hairpin) was replaced by a His-tag (E-Syt1cyto, Fig 1A), was added to the mixture of two populations of liposomes. One liposome population (anchoring liposomes, ER-like in composition) comprised phosphatidylcholine (PC), a nickel-conjugated lipid [DGS-NTA(Ni)] that functions as a binding site for the His-tagged proteins, and NBD-phosphatidylethanolamine (PE). The other liposome population (target liposomes, PM-like in composition) comprised PC and two acidic phospholipids, phosphatidylserine (PS) and PI(4,5)P2. The increase in turbidity, which reflects clustering of liposomes into larger particles, was measured as optical density at 405 nm. Addition to the liposome mixture of E-Syt1cyto in a buffer devoid of Ca2+ resulted in an increase in optical density (turbidity; Fig 1B). Upon addition of a “cocktail” of EGTA, imidazole (to disrupt the nickel His-tag interaction) and proteinase K at the end of incubation, the increase in optical density was reversed, ruling out the fusion of liposomes as the cause of the increase in optical density (Fig 1B). No change in optical density was observed in the absence of E-Syt1cyto, of DGS-NTA(Ni) in the anchoring liposomes, or of the target liposomes (Fig 1B). We conclude that although E-Syt1 is primarily diffuse throughout the ER when expressed alone, under in vitro conditions, it can tether ER-like to PM-like liposomes even in the absence of Ca2+. Actually, low level of E-Syt1-dependent ER-PM contact sites can be observed at resting Ca2+ concentration in the cells (Giordano et al, 2013; Fernandez-Busnadiego et al, 2015). It was shown that the C2E domain of E-Syt1 shares the properties of the C2C domains of E-Syt2 and E-Syt3, which in these two proteins mediate constitutive PI(4,5)P2-dependent PM binding (Giordano et al, 2013; Fernandez-Busnadiego et al, 2015; Idevall-Hagren et al, 2015). All these three C-terminal C2 domains lack a Ca2+-binding site and have a highly basic surface in common (Idevall-Hagren et al, 2015). Accordingly, and consistently with studies in cells (Fernandez-Busnadiego et al, 2015; Idevall-Hagren et al, 2015), a mutant E-Syt1cyto that lacks the C2E domain (SMP-C2ABCD, Fig 1A) failed to tether target liposomes in the absence of Ca2+ (Fig 1B). Lack of PI(4,5)P2 in the target liposomes, or replacement of the 5% PI(4,5)P2 with 20% PS in these liposomes, also dramatically reduced E-Syt1cyto-mediated Ca2+-independent membrane tethering (Fig 1B). These results indicate that in the liposome-based system, the cytosolic domain of E-Syt1 mediates tethering of the two classes of liposomes in a C2E-dependent way in the absence of Ca2+. We next investigated the role of Ca2+ in liposome tethering using the same liposome-based assay. Addition of Ca2+ (100 μM) resulted in a strong increase in the basal tethering by E-Syt1cyto observed in the Ca2+-free buffer (Fig 1C). E-Syt1 contains putative Ca2+-binding sites in its C2A and C2C domains (Min et al, 2007; Giordano et al, 2013; Xu et al, 2014). Mutations of key residues that mediate Ca2+ binding in the C2C domain of the construct (E-Syt1cyto C2Cx, Fig 1A) only showed a slightly increased Ca2+-dependent membrane tethering (Fig 1C). In contrast, mutations in the Ca2+-binding sites in the C2A domain (E-Syt1cyto C2Ax, Fig 1A) still produced a significant increase in Ca2+-dependent liposome tethering (Fig 1C). Both of these mutants had a similar level of Ca2+-independent basal liposome tethering (Fig 1C). These findings were consistent with the previous reports that the C2C domain is required for Ca2+-dependent ER-PM tethering of E-Syt1 in intact cells (Chang et al, 2013; Giordano et al, 2013). A lower degree of Ca2+-dependent tethering occurred also with an E-Syt1cyto fragment lacking the C2E domain (SMP-C2ABCD, Fig 1A and D). This tethering did not require PI(4,5)P2 in target liposomes (Fig 1D) and occurred at a similar level for both constructs harboring Ca2+-binding mutations in either the C2A domain (SMP-C2AxBCD) or the C2C domain (SMP-C2ABCxD; Fig EV1A), although binding of SMP-C2ABCxD occurred with slower kinetics (Fig EV1B). In addition, a similar dependence on Ca2+ concentration [half maximal effective concentration (EC50)] was observed for the binding of SMP-C2AxBCD and SMP-C2ABCxD to membranes, as revealed by the liposome tethering assay (Fig EV1D). Given that E-Syt1cyto C2Ax showed a much stronger Ca2+-dependent stimulation of tethering than E-Syt1cyto C2Cx (Fig 1C), and C2E does not bind Ca2+, we conclude that the increased E-Syt1cyto-mediated liposome tethering in the presence of Ca2+ mainly relies on a synergistic effect between Ca2+-bound C2C and C2E. This most likely occurs via the release of an inhibitory action of C2C affecting C2E binding to the PI(4,5)P2-rich membranes, since an E-Syt1 construct lacking the C2ABCD region (SMP-C2E, Fig EV1A) tethered the liposomes at a higher level than E-Syt1cyto in the absence of Ca2+ (Fig EV1C). This inhibition did not completely abolish the membrane binding of C2E in the absence of Ca2+, as C2E-dependent and Ca2+-independent liposome tethering by E-Syt1cyto could be observed (Fig 1B). Click here to expand this figure. Figure EV1. Ca2+ binding to C2C promotes E-Syt1cyto-mediated liposome tethering by releasing an inhibitory action of C2C on the membrane binding of C2E A. Domain structures of E-Syt1 constructs used for the liposomes tethering assays shown in (B and C). Asterisks indicate the mutations in the membrane binding surface of C2A domain. B, C. Tethering of anchoring and target liposomes in the presence of E-Syt1 constructs at RT as assessed by increase in turbidity (OD 405 nm). In each of the panels, time-courses are at left and bar graphs showing quantification of OD405 increases at the end of the incubation (arrows in the left panels) are at right. (B) Effect of the absence or presence of Ca2+ on liposome tethering by SMP-C2AxBCD, SMP-C2ABCxD, and SMP-C2AmBCxD. Mean and SD of three independent experiments. P-values from t-test with Bonferroni corrections are quoted on the graphs. ****P < 0.0001; n.s. not significant. (C) Liposome tethering by E-Syt1cyto with or without C2ABCD domains in the absence of Ca2+. Mean and SD of three independent experiments. P-values from two-tailed Student's t-test are quoted on the graphs. ***P < 0.001. D. SMP-C2ABCxD and SMP-C2AxBCD bind to liposomes in a Ca2+-dependent manner, as revealed by liposome tethering. OD405 readings were normalized to the maximum value. Mean and SD of three independent experiments. E. Left: Purified C2CD and C2E were incubated in the presence of the cross-linker BS3 and in the absence or presence of Ca2+ and liposomes. The left lane shows molecular weight markers, with sizes indicated in kilodaltons. The cross-linked heterodimer, as confrmed by mass spectrometry, is indicated by the red arrowhead. Right: Normalized intensity of cross-linked heterodimer was plotted. Mean and SD of three independent experiments. P-values from t-test with Bonferroni corrections are quoted on the graphs. **P < 0.01; n.s., not significant. F. Sequence of C2CD and C2E used for cross-linking assay. The peptides identified by mass spectrometry are highlighted in red. Download figure Download PowerPoint To confirm that the inhibitory action of C2C on C2E is through a direct interaction, we purified C2CD and C2E, respectively, and performed cross-linking experiments. After incubation with the cross-linker BS3 [bis(sulfosuccinimidyl) suberate], a band with MW corresponding to that expected for a C2CD-C2E heterodimer appeared (Fig EV1E, red arrowhead, lanes 6, 7, and 8). The heterodimer of C2CD-C2E was confirmed by mass spectrometry analysis of the band, which reveled peptides of both C2CD and C2E, covering nearly their entire sequences (Fig EV1F). Importantly, formation of this band in response to the cross-linker was strongly reduced when the two protein fragments were incubated with both Ca2+ and liposomes, that is, when C2CD can bind to membranes (lane 9, see right panel for quantification). The band was only weak, but this was not unexpected as in the intact protein the C2CD and the C2E domain are part of the same polypeptide and thus are in close proximity (which will increase the efficiency of heterodimer formation), while in our case, they are dispersed in solution. This result strongly supports the hypothesis that C2CD inhibits the binding of C2E to a PI(4,5)P2-containing membrane through a direct interaction, which will be released by Ca2+ binding to C2C to redirect it to the membrane. Altogether, these results demonstrate that both PI(4,5)P2 binding by C2E and Ca2+ regulation of C2C play the dominant role in the Ca2+ stimulation of E-Syt1-dependent liposome tethering. C2A domain of E-Syt1 participates in the Ca2+ regulation of lipid transfer in vitro In view of the lack of a relevant impact of the Ca2+-binding properties of C2A on Ca2+-dependent liposome tethering by E-Syt1 (Fig 1C), we explored whether these properties play a role in lipid transfer using a fluorescence resonance energy transfer (FRET)-based assay (Saheki et al, 2016; Yu et al, 2016). The assay involves the same liposomes described above (with the addition of NBD-PE to the anchoring liposomes, henceforth defined as donor liposomes). The fluorescence of NBD, which is partially self-quenched in these liposomes, increases as a result of dequenching since NBD-PE is transferred to the target liposomes (henceforth defined as acceptor liposomes). Consistent with previous reports (Saheki et al, 2016; Yu et al, 2016), Ca2+ strongly stimulated lipid transfer by E-Syt1cyto, as revealed by NBD-PE dequenching (Fig 2B). However, this stimulation was completely abolished by the Ca2+-binding mutations in C2A (E-Syt1cyto C2Ax), in spite of very little impact of these mutations on liposome tethering (Fig 2B). Figure 2. Ca2+ binding to C2A is required for lipid transfer by the SMP domain when flanked by the C2AB domain A. Domain structures of constructs used for the lipid transfer assays shown in the figure. PH = PH domain of rat PLCδ. Slanted white lines indicate the mutations in the Ca2+ binding sites in C2A and C2C domains. Asterisks indicate the mutations in the membrane binding surface of C2A domain. B. Left: Time-course of normalized fluorescence signals of liposomes mixtures containing 1.5% NBD-PE in the donor liposomes in the absence or presence of Ca2+ at RT. E-Syt1 constructs were added at time 0 (protein:lipid ratio 1:1,000). Right: Quantification of the increase in NBD fluorescence at the end of the incubation (arrow in left). C–E. Lipid transfer between donor and acceptor liposomes in the presence of E-Syt1 constructs (protein:lipid ratio 1:400) at 37°C as assessed by dequenching of NBD-PE fluorescence. In each of the panels, time-courses are at the top and bargraphs showing quantification of NBD fluorescence at the end of the incubation (arrows in the upper panels) are at the bottom. (C) Effect of C2ABCDE-dependent liposome tethering on lipid transfer mediated by the SMP domain. (D) Effect of the Ca2+-binding property of C2A on the lipid transfer activity of the SMP domain between C2ABCDE-tethered liposomes. (E) Effect of Ca2+ on the lipid transfer activity of the SMP domain alone or SMP-C2AB on liposomes tethered by a PH domain. Data information: Mean and SD of three independent experiments. P-values from t-test with Bonferroni corrections are quoted on the graphs. **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. Download figure Download PowerPoint SMP domain of E-Syt1 alone can transfer lipids in a Ca2+-independent manner between tethered membranes To investigate the interplay of the SMP domain and C2 domains in SMP domain-dependent lipid transfer, His-tagged SMP domain of human E-Syt1 (SMP, Fig 2A) was generated and tested for its activity in the in vitro lipid transfer assay. No increase in turbidity (consistent with lack of tethering; Fig EV2A) or in lipid transfer was observed upon incubation of the SMP domain alone with donor and acceptor liposomes, even in the presence of Ca2+ (Fig 2C). While in principle even the SMP domain alone could mediate some lipid transfer during random encounters between liposomes, the rate of such transfer may be too low to be detected during the assay period (30 min) in the absence of tethering. However, when both SMP and a His-tagged C2ABCDE fragment of E-Syt1 (C2ABCDE, Fig 2A) were added together to the mixtures of donor and acceptor liposomes, SMP domain-dependent lipid transfer was observed in the presence of Ca2+ (Fig 2C), that is, conditions under which the C2ABDCE can mediate tethering (Fig EV2A). Ca2+ appeared to be needed only to facilitate tethering, as a similar degree of lipid transfer by SMP domain was observed irrespective of the presence of Ca2+, when the two sets of liposomes were connected by another tether, His-tagged PHPLCδ, which binds to PI(4,5)P2 (Garcia et al, 1995; Hammond & Balla, 2015) in the acceptor liposomes in a Ca2+-independent way (Figs 2E and EV2B). These experiments further demonstrate that a key role of Ca2+ in E-Syt1-dependent lipid transfer is to mediate tethering. Additionally, these results also reveal that the additional importance of Ca2+ binding to the C2A domain in enabling lipid transfer is only manifested when the SMP and C2A are part of the same polypeptide. Click here to expand this figure. Figure EV2. Both C2ABCDE and PHPLCδ can tether liposomes, although C2ABCDE, but not PHPLCδ requires Ca2+ A, B. Tethering of anchoring and target liposomes in the presence of the constructs indicated (see Fig 4A, protein:lipid ratio 1:400) at 37°C as assessed by increase in turbidity (OD 405 nm). In each of the panels, time-courses are at left and bar graphs showing quantification of OD405 increases at the end of the incubation (arrows in the left panels) are at right. (A) Effect of the absence or presence of Ca2+ on liposome tethering by SMP or C2ABCDE. Mean and SD of three independent experiments. P-values from t-test with Bonferroni corrections are quoted on the graphs. ****P < 0.0001; n.s. not significant. (B) Effect of the absence or presence of Ca2+ on liposome tethering by PHPLCδ. Mean and SD of three independent experiments. P-values from two-tailed student's t-test are quoted on the graphs. n.s. not significant. Download figure Download PowerPoint C2A domain of E-Syt1 inhibits the activity of SMP domain in the absence of Ca2+ via an intramolecular interaction To further analyze the property of the C2A domain of E-Syt1 in lipid transfer, His-tagged SMP-C2AB and His-tagged SMP domain were tested in parallel. When added to donor and acceptor liposomes, no transfer was observed with the SMP domain alone (see above, Figs 2C and EV3A), while the SMP-C2AB construct induced some liposome tethering and lipid transfer, but only in the presence of Ca2+ (Fig EV3). The lipid transfer properties of the SMP-C2AB fragment were much more pronounced, and only in the presence of Ca2+, when liposomes were additionally tethered by the His-tagged C2ABCDE fragment (Fig 2D). However, mutations in C2A domain that make SMP-C2AB incapable of binding Ca2+ (SMP-C2AxB, Fig 2A) abolished its lipid transfer activity even in the presence of Ca2+ and of C2ABCDE (Fig 2D). Under these conditions (presence of C2ABCDE and Ca2+), a robust lipid transfer was achieved by SMP alone (see above, Fig 2C and D), although it was lower than SMP-C2AB (Fig 2D), which is possibly due to a higher basal lipid transfer by SMP-C2AB compared to SMP alone in the presence of Ca2+ (Fig EV3A). Click here to expand this