Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII.

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.


INTRODUCTION
Neurotransmitter release is achieved by the fusion of docked synaptic vesicles with the presynaptic plasma membrane in response to a rapid increase in intracellular Ca 2+ concentrations (up to about 200 µM) due to the opening of voltage-gated Ca 2+ channels (1,2). Ca 2+ -binding proteins on the synaptic vesicles are required to sense this rapid increase in Ca 2+ ions. Recent genetic and biochemical studies have indicated that synaptotagmin I (Syt I) 1 , a Ca 2+ , phospholipid, and inositol polyphosphate binding protein present on synaptic vesicles, is the best candidate for the Ca 2+ -sensor of neurotransmitter release in the central nervous system (reviewed in Refs. [3][4][5][6]. The synaptotagmins now constitute a large protein family with twelve isoforms described in rat or mouse (Ref. 7 and references therein), and are thought to be involved in vesicular trafficking. Each member shares a short amino terminus, a single transmembrane region, and two C2 domains (known as the C2A and C2B domains) homologous to the C2 regulatory region of protein kinase C (reviewed in Refs. [3][4][5][6]. Syt I, the best characterized isoform of synaptotagmin, is thought to function in an oligomerized state on the basis of genetic analyses of Drosophila synaptotagmin mutants (reviewed in Ref. 8) and in vitro biochemical studies (9)(10)(11)(12)(13). Syt I (or II) has two selfoligomerization properties: A SDS-resistant Ca 2+ -independent multimerization probably mediated by a region just downstream of the transmembrane region (7,14,15) and a Ca 2+ -dependent oligomerization mediated by the C2B domain (9)(10)(11)(12)(13). However, the functional relationship between these two interactions remains unknown. For instance, it remains undetermined whether the C2B domains oligomerize independent of the Ca 2+independent oligomerization at the amino (N)-terminal domain (Fig. 1A). In addition, studies of the Ca 2+ -dependent oligomerization of the C2B domain have mainly used recombinant proteins from Escherichia coli (9)(10)(11)(12)(13), which can be very sticky compared to native proteins prepared from mouse brain, and tend to aggregate in low Ca 2+ ion concentrations 2 . Studies of Ca 2+ -independent oligomerization using recombinant proteins from E. coli may also be unreliable because native synaptotagmins undergo post-by guest on July 23, 2018 http://www.jbc.org/ Downloaded from 4 translational modifications such as palmitoylation (16,17) or disulfide bond formation (7). Thus, it would be better to examine the oligomerization properties of the synaptotagmin family in mammalian cultured cells by expressing two different epitope (FLAG and T7)-tagged synaptotagmins (7). In this method, we previously showed that Syts III, V, VI and X form homo-and hetero-oligomer through the N-terminal cysteine motif by disulfide bonding (at amino acids 10, 21, and 33 of mouse synaptotagmin III) irrespective of the presence of Ca 2+ (7), and that Syt VI∆TM, a major alternatively spliced variant of Syt VI that lacks this cysteine motif, cannot associate with Syt VI in vitro or in vivo (18). In contrast, other isoforms did not show such cysteine-based homooligomerization (7).
In the present study, we examined the Ca 2+ -dependent and -independent oligomerization properties of synaptotagmin isoforms (both full length and cytoplasmic portion) using two different epitope-tagged synaptotagmins, and found that Syt VII is the only isoform that shows robust Ca 2+ -dependent oligomerization properties. On the basis of these results, we discuss the functional relationship between Ca 2+ -dependent andindependent oligomerization of synaptotagmins.

Construction of T7-and FLAG-tagged Synaptotagmin I-XI Cytoplasmic Domains
Construction of the full cytoplasmic domains of mouse synaptotagmins I-XI were carried out by polymerase chain reaction using the following oligonucleotides with primers using pGEM-T-FLAG-Syt I (7) as a template. Purified PCR products were digested with BamHI and SalI and then substituted for the BamHI-SalI insert of pGEM-T-FLAG-Syt I, and verified by DNA sequencing as described above. The resulting pGEM-T-FLAG-Syt I-cyto plasmid was digested with NotI, and then the NotI insert was subcloned into the NotI site of pEF-BOS (pEF-FLAG-Syt I-cyto) (19,20). The pEF-FLAG-Syts II-XI-cyto clones were similarly constructed. pEF-T7(or FLAG)-Syts I-XI were prepared as described previously (7). Plasmid DNA was prepared using Wizardmini preps (Promega) or QIAGEN Maxi prep kits.

Data Processing
Statistical analysis and curve fitting were done using the GraphPad PRISM computer program (Version 2.0).

Ca 2+ -dependent and -independent Self-oligomerization of Synaptotagmin Isoforms
In a previous study, we showed that Syts III, V, VI, and X formed heterooligomers through disulfide bonding irrespective of the presence of Ca 2+ (7).  1C). Based on the differences in homo-oligomerization activities, we classify these isoforms into three distinct groups. The first group included Syts I, II, VIII, and XI, which showed Ca 2+independent homo-oligomerization activity, but Syt XI oligomerization activity was apparently weaker than that of the others (Syts I, II, and VIII). Among this group, only Syt II oligomerization was slightly activated by Ca 2+ (<1.5-fold increase; Fig. 1C). The second group contained Syts IV and IX. Syt IV did not form homo-oligomers in our binding conditions, and faint Syt IX homo-oligomerization was only observed after prolonged exposure to X-ray film. Consistent with this, we previously showed that these isoforms did not form significant SDS-resistant homo-oligomers on SDS-PAGE (7). The

Ca 2+ -dependent Oligomerization Properties of Cytoplasmic Domains from Synaptotagmins I-XI
In the next set of experiments, we examined the Ca 2+ -dependent oligomerization properties of synaptotagmin cytoplasmic domains as demonstrated by the interaction of recombinant Syt I or II expressed in bacteria with detergent-solubilized native proteins (Syt I) from brain (9)(10)(11)(12)(13). Consistent with the results shown in Fig. 1 of which belong to the same subclass of synaptotagmins (7,21), in a Ca 2+ -dependent manner ( Fig. 3 and data not shown). Syts V, VI, and X were found to heterooligomerize as well with Syt VII as Ca 2+ -dependent Syt VII homo-oligomerization.
Similar results were obtained even when the full length Syts III, V, VI, VII, and X were used (data not shown).

Properties of Synaptotagmins
Since the homo-oligomerization of full length and the cytoplasmic domain of Syt VII were strongly activated by Ca 2+ (Fig. 1B and Fig. 2), Ca 2+ -dependent oligomerization of the Syt VII cytoplasmic domain would occur between two molecules that do not preassemble at the N-terminal domain (see Fig. 1A, right panel). This was evident when three proteins (T7-Syt VII, FLAG-Syt VII, and FLAG-Syt VII-cyto; molar ratio, 1:1:1) were co-expressed in COS-7 cells (Fig. 4B, lane 2) and their associations were analyzed by immunoprecipitation. In the absence of Ca 2+ , only FLAG-Syt VII weakly interacted with T7-Syt VII (Fig. 4A, lane 3), whereas in the presence of Ca 2+ , equivalent amounts of Syt VII cytoplasmic domain (FLAG-Syt VII-cyto) and Flag-Syt VII were coimmunoprecipitated with T7-Syt VII (Fig. 4A,  In contrast, when T7-Syt II, FLAG-Syt II, and FLAG-Syt II-cyto were coexpressed in COS-7 cells (molar ratio, 1:1:1), the Syt II cytoplasmic domain (FLAG-Syt II-cyto) could not interact with full length proteins (T7-Syt II) even in the presence of Ca 2+ (Fig. 4A, lanes 1 and 2), even though the Syt II cytoplasmic domain itself showed Ca 2+ -dependent oligomerization (Fig. 2). However, FLAG-Syt II could oligomerize well with T7-Syt II irrespective of the presence of Ca 2+ , as demonstrated in Fig. 1A and 1B.   Fig. 5C and data not shown), suggesting that these associations are physiologically relevant.

DISCUSSION
In this study, we have demonstrated that synaptotagmin isoforms show distinct homo-oligomerization activities by using a dual epitope-tag (T7 and FLAG) expression system. Based on the results shown in Fig. 1B, together with the previously described cysteine-based oligomerization of Syts III, V, VI, and X (7), we classified the synaptotagmin family into four distinct groups. The first group contained Syts III, V, VI, and X, which were strongly associated with each other via the conserved N-terminal cysteine motif by disulfide bonding (7), and accordingly their associations were βmercaptoethanol-sensitive and Ca 2+ -independent. At the cytoplasmic domain, however, only Syt V cytoplasmic domain showed apparent Ca 2+ -dependent homo-oligomerization ( Fig. 2). This grouping is consistent with a previous phylogenetic analysis (7) and their reported weak or no inositol 1,3,4,5-tetrakisphosphate binding ability (21).
The second group (Syts I, II, VIII, and XI) also showed Ca 2+ -independent homooligomerization, but these interactions were insensitive to β-mercaptoethanol (7). Syt XI homo-oligomerization activity was significantly lower than those of Syts I, II, and VIII.
Despite the high sequence similarity between Syts I and II (7) Moreover, previous in vitro binding experiments only showed the existence of Ca 2+dependent oligomerization, but did not address the strength (e.g. K d value) of the Syt I oligomerization (9)(10)(11)(12)(13). Further work will be required to elucidate whether the Ca 2+dependent oligomerizing capacity of Syt I occurs at physiologically relevant levels. Syt I 12 Ca 2+ -independent oligomerization is thought to be mediated by the predicted amphipathic α-helix region just downstream of the transmembrane region (14). However, since Syts II and VIII did not contain such amphipathic α-helix regions and only the Syt II cytoplasmic domain showed Ca 2+ -independent oligomerization (Fig. 2) (Fig. 1A, left panel). In addition, we found that Syt VII can completely heterooligomerize with Syts V, VI, and X, but not Syt III, in a Ca 2+ -dependent manner. Since Syts III, V, VI and X can be assembled by disulfide bonding at the N-terminal domain (7), Syt VII binds to the cytoplasmic domain of Syts V, VI, and X in response to Ca 2+ , which would modulate the function of the Syts III, V, VI and X complexes. Since Syts III, VI, and VII are expressed in all brain regions and Syts V and X are brain-specific isoforms (25)(26)(27)(28), at least four isoforms are co-expressed in the same brain regions.
Thus, it is possible to form such Ca 2+ -dependent hetero-oligomers. Currently, however, except for the function of Syt III in insulin secretion from pancreatic β-cells (29), the exact role of Syts III, V, VI, and X in brain remains obscure.
Recently, Chapman et al. reported (12) the C2B effector domain of Syt I (Lys at positions 326 and 327 (K326, and K327)), which is crucial for self-oligomerization (9)(10)(11)(12)(13), and for binding to inositol 1,3,4,5-tetrakisphosphate (22,30), AP-2 (25,31,32) and the synprint region of the α1B subunit of N-type Ca 2+ channels (33). Although these two Lys residues in the C2B domain are well conserved in almost all the isoforms (7,25), Ca 2+ -dependent homo-oligomerization activities were apparently different between each isoform (Fig. 2), suggesting that the surrounding sequences would affect and 13 determine homo-oligomerization capacity. In fact, the Syt VII C2B domain has several unique amino acid residues that are not conserved in other isofomrs (e.g. I284, N328, S373 e.t.c.). Further detailed mutational analysis is needed to elucidate the structural basis of Ca 2+ -dependent oligomerization of the cytoplasmic domain of Syt VII in the future. In contrast, when the C2B domains of Syts III, V, VI, and X were compared, we could not identify specific amino acid residues unique to Syt III, which are implicated in loss of Ca 2+ -dependent oligomerization with Syt VII.
In summary, we have investigated the Ca 2+ -dependent and -independent homooligomerization of synaptotagmin family proteins and show that the Ca 2+ -independent oligomerization at the N-terminal domain is a prerequisite for the Ca 2+ -dependent oligomerization at the cytoplasmic domain of synaptotagmin family proteins (except for Syt VII). Therefore, studies on the Ca 2+ -dependent hetero-oligomerization at the cytoplasmic domain will have to be evaluated by using both full length and cytoplasmic domains. Ca 2+ -dependent and -independent hetero-oligomerization of the synaptotagmin family will be clarified by the T7-and FLAG-tagged Syts co-expression system in the near future.