Mechanism of the SDS-resistant Synaptotagmin Clustering Mediated by the Cysteine Cluster at the Interface between the Transmembrane and Spacer Domains

• Published in: The Journal of biological chemistry Vol. 276; no. 43; pp. 40319 - 40325
• Main Authors: Fukuda, Mitsunori, Kanno, Eiko, Ogata, Yukie, Mikoshiba, Katsuhiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.10.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Calcium-Binding Proteins - metabolism; Cysteine; Membrane Glycoproteins - metabolism; Mice; Molecular Sequence Data; Nerve Tissue Proteins - metabolism; Protein Structure, Quaternary; Receptor Aggregation - drug effects; Receptors, Cell Surface - metabolism; Signal Transduction; Sodium Dodecyl Sulfate - pharmacology; Synaptotagmin I; Synaptotagmins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M105356200

Synaptotagmin I (Syt I), a proposed major Ca2+ sensor in the central nervous system, has been hypothesized as functioning in an oligomerized state during neurotransmitter release. We previously showed that Syts I, II, VII, and VIII form a stable SDS-resistant, β-mercaptoethanol-insensitive, and Ca2+-independent oligomer surrounding the transmembrane domain (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180–28185), but little is known about the molecular mechanism of the Ca2+-independent oligomerization by the synaptotagmin family. In this study, we analyzed the Ca2+-independent oligomerization properties of Syt I and found that it shows two distinct forms of self-oligomerization activity: stable SDS-resistant self-oligomerization activity and relatively unstable SDS-sensitive self-oligomerization activity. The former was found to be mediated by a post-translationally modified (i.e. fatty-acylated) cysteine (Cys) cluster (Cys-74, Cys-75, Cys-77, Cys-79, and Cys-82) at the interface between the transmembrane and spacer domains of Syt I. We also show that the number of Cys residues at the interface between the transmembrane and spacer domains determines the SDS- resistant oligomerizing capacity of each synaptotagmin isoform: Syt II, which contains seven Cys residues, showed the strongest SDS-resistant oligomerizing activity in the synaptotagmin family, whereas Syt XII, which has no Cys residues, did not form any SDS-resistant oligomers. The latter SDS-sensitive self-oligomerization of Syt I is mediated by the spacer domain, because deletion of the whole spacer domain, including the Cys cluster, abolished it, whereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomerization of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys cluster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.