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. 2005 Apr 5;102(14):5210-4.
doi: 10.1073/pnas.0500941102. Epub 2005 Mar 25.

Three distinct kinetic groupings of the synaptotagmin family: candidate sensors for rapid and delayed exocytosis

Enfu Hui  1 , Jihong Bai, Ping Wang, Mutsuyuki Sugimori, Rodolfo R Llinas, Edwin R Chapman
Affiliations

Three distinct kinetic groupings of the synaptotagmin family: candidate sensors for rapid and delayed exocytosis

Enfu Hui et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

Synaptotagmins (syts) are a family of membrane proteins present on a variety of intracellular organelles. In vertebrates, 16 isoforms of syt have been identified. The most abundant isoform, syt I, appears to function as a Ca2+ sensor that triggers the rapid exocytosis of synaptic vesicles from neurons. The functions of the remaining syt isoforms are less well understood. The cytoplasmic domain of syt I binds membranes in response to Ca2+, and this interaction has been proposed to play a key role in secretion. Here, we tested the Ca(2+)-triggered membrane-binding activity of the cytoplasmic domains of syts I-XII; eight isoforms tightly bound to liposomes that contained phosphatidylserine as a function of the concentration of Ca2+. We then compared the disassembly kinetics of Ca2+.syt.membrane complexes upon rapid mixing with excess Ca2+ chelator and found that syts can be classified into three distinct kinetic groups. syts I, II, and III constitute the fast group; syts V, VI, IX, and X make up the medium group; and syt VII exhibits the slowest kinetics of disassembly. Thus, isoforms of syt, which have much slower disassembly kinetics than does syt I, might function as Ca2+ sensors for asynchronous release, which occurs after Ca2+ domains have collapsed. We also compared the temperature dependence of Ca2+.syt.membrane assembly and disassembly reactions by using squid and rat syt I. These results indicate that syts have diverged to release Ca2+ and membranes with distinct kinetics.

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Figures

Fig. 1.
Fig. 1.
Interaction of syt isoforms with PS/PC liposomes. (A) Molecular model depicting the cytoplasmic domain (containing the C2A and C2B domains) of syt. The crystal structure of the cytoplasmic domain of syt III (45) was used as the template, and the membrane anchor and lipid bilayer were added by using a drawing program. For all experiments described in this study, syt refers to the intact cytoplasmic domain lacking the membrane-anchoring domain. (B) Screening syts I–XII for Ca2+-triggered PS/PC liposome-binding activity. syt·PS/PC liposome interactions were studied by using fluorescently labeled liposome pull-down assay. The cytoplasmic domains of syts I–XII were immobilized on glutathione-Sepharose beads and assayed for binding of PS/PC liposome in the presence of 2 mM EGTA (open bars) or 0.2 mM Ca2+ (filled bars), as described in Materials and Methods. Liposomes were composed of 1% N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 25% PS, and 74% PC. (C) syt III exhibits a higher apparent affinity for Ca2+ than does syt I or IX. Immobilized GST-syt I, GST-syt III, and GST-syt IX were assayed for 3H-labeled liposome (25% PS/75% PC)-binding activity as a function of [Ca2+] as described in ref. . The [Ca2+]1/2 values for syts I, III, and IX were 18.8 ± 2.2, 7.0 ± 0.8, and 13.6 ± 1.3 μM, respectively; the Hill slopes for syts I, III, and IX were 2.0 ± 0.3, 2.3 ± 0.3, and 3.5 ± 0.3, respectively. (D) Model describing the assembly of Ca2+·syt·liposome complexes. The rate constants measured in these experiments correspond to the on-rate (kon) and off-rate (koff) for the second step; Ca2+ binding in the first step is too fast to measure in these experiments because of the need to use micromolar levels of Ca2+ to trigger binding (29). (E and F) Comparison of the on- and off-rates for interactions of syts I, III, and IX with PS/PC liposomes in the presence of Ca2+. Stopped-flow rapid-mixing experiments were carried out at 14.5°C as described in Materials and Methods. (E) Representative kinetics traces of syts I, III, and IX are shown. Liposomes (22 nM, 5% dansyl-PE/25% PS/70% PC) were premixed with 0.2 mM Ca2+ and then rapidly mixed (1:1) with 4 μM syt I, III, or IX. Single exponential functions were used to determine the observed rates (kobs) of Ca2+-dependent syt I and syt III·liposome interactions; a double exponential was needed to fit the data for syt IX·liposome interactions. (F) kobs was plotted as a function of [liposome]. In the case of syt IX, the fast component was used. The y intercept yields koff, and the slope yields kon for the interaction of syt with membranes in the presence of Ca2+. Error bars represent SDs of three independent experiments. The dissociation constants (Kd) for syt·liposome interactions in the presence of Ca2+ were calculated as koff/kon. Values of Kd, kon, and koff are summarized in Table 1. Note that the rate constants of the slower component for syt IX·liposomes interactions were not dependent on the [liposome], suggesting that this component reports a postbinding conformational change in syt IX.
Fig. 2.
Fig. 2.
syts are released from liposomes upon chelation of Ca2+ with distinct kinetics. Disassembly of Ca2+·syt·liposome complexes was monitored by using FRET as shown in Fig. 1E. All experiments were carried out at 14.5°C. Liposomes (44 nM) were premixed with syt (4 μM) in the presence of Ca2+ (0.2 mM) and then rapidly mixed with an equal volume of Hepes buffer containing 2 mM EGTA. The rate constants for disassembly (kdiss), determined by fitting the kinetics traces with single or double exponential functions, are provided in Table 2. (A) Model depicting the disassembly of Ca2+·syt·liposome complexes upon rapid mixing with EGTA. (B) Representative traces of disassembly reactions. Based on the disassembly kinetics, syt isoforms are divided into three kinetic groups: fast, medium, and slow. (C) syts I, II, and III exhibit fast disassembly kinetics. (D) syts IX, X, V, and VI disassemble from liposomes at intermediate rates. (E) syt VII disassembles from liposomes with the slowest kinetics.
Fig. 3.
Fig. 3.
The temperature dependence of the kinetics of rat and squid syt I interactions with PS/PC liposomes. For these experiments, FRET was used to monitor the time course of assembly (kobs) and disassembly (kdiss) of syt·liposome complexes, as shown in Fig. 1E. (A) Temperature dependence of the kinetics of Ca2+-triggered syt·liposome interactions. (Left) Representative kinetics traces of squid and rat syt I binding to liposomes in response to Ca2+ at 25°C. The FRET signal for rat Ca2+·syt·liposome assembly was a single exponential. The traces obtained by using squid syt were best fitted by double exponentials; the slower component might represent postbinding conformational changes or molecular rearrangements [e.g., oligomerization (35)] because it is not dependent on [liposome]. (Right) Arrhenius plot of ln(kobs) versus 1/T for the interaction of rat and squid (the fast component) syt with liposomes. The activation energy (Ea) was calculated from the slope, and this value was 15.8 ± 1.4 kJ/mol for squid syt I and 13.5 ± 1.2 kJ/mol for rat syt I. (B) Temperature dependence of the disassembly kinetics of syt·liposome complexes upon removal of Ca2+. (Left) Kinetic traces of squid and rat syt I unbinding from liposomes upon rapid mixing with excess EGTA at 10°C. Disassembly traces were well fitted with single exponential functions. The disassembly of squid syt·liposome complexes is much slower than that for the complex containing rat syt I. (Right) Arrhenius plot of ln(kdiss) versus 1/T for the disassembly of rat and squid (the fast component) syt I from liposomes. Ea was 42.1 ± 1.2 kJ/mol for squid syt I and 29.9 ± 2.2 kJ/mol for rat syt I. The disassembly of rat syt·liposome complexes was too fast to monitor when at temperatures > 25°C (i.e., the reaction was complete within the 1-msec dead time of the stopped-flow setup).
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