Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains

Synaptotagmin I (Syt I), a proposed major Ca2+ sensor in the central nervou s system, has been hypothesized as functioning in an oligomerized state dur ing neurotransmitter release. We previously showed that Syts I, II, VII, an d VIII form a stable SDS-resistant, beta -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 kn own about the molecular mechanism of the Ca2+-independent oligomerization b y the synaptotagmin family. In this study, we analyzed the Ca2+-independent oligomerization properties of Syt I and found that it shows two distinct f orms of self-oligomerization activity: stable SDS-resistant self-oligomeriz ation activity and relatively unstable SDS-sensitive self-oligomerization a ctivity. The former was found to be mediated by a post-translationally modi fied (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 d omains of Syt I. We also show that the number of Cys residues at the interf ace between the transmembrane and spacer domains determines the SDS-resista nt oligomerizing capacity of each synaptotagmin isoform: Syt II, which cont ains seven Cys residues, showed the strongest SDS-resistant oligomerizing a ctivity in the synaptotagmin family, whereas Syt XII, which has no Cys resi dues, did not form any SDS-resistant oligomers. The latter SDS-sensitive se lf-oligomerization of Syt I is mediated by the spacer domain, because delet ion of the whole spacer domain, including the Cys cluster, abolished it, wh ereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomeri zation of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys c luster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.