Calcium-dependent and -independent hetero-oligomerization in the synaptotagmin family

Synaptotagmins constitute a family of membrane proteins that are characterized by one transmembrane region and two C2 domains. Recent genetic and biochemical studies have indicated that oligomerization of synaptotagmin (Syt) I is important for expression of function during exocytosis of synaptic vesicles. However, little is known about hetero-oligomerization in the synaptotagmin family. In this study, we showed that the synaptotagmin family is a type I membrane protein (N-lumen/C-cytoplasm) by introducing an artificial N-glycosylation site at the N-terminal domain, and systematically examined all the possible combinations of hetero-oligomerization among synaptotagmin family proteins (Syts I-XI), We classified the synaptotagmin family into four distinct groups based on differences in Ca2+-dependent and -independent oligomerization activity. Group A Syts (III, V,VI, and X) form strong homo- and hetero-oligomers by disulfide bonds at an N-terminal cysteine motif irrespective of the presence of Ca2+ [Fukuda, M., Kanno, E,, and Mikoshiba, K (1999) J. Biol. Chem. 274, 31421-31427], Group B Syts (I, II, VIII, and M) show moderate homo-oligomerization irrespective of the presence of Ca2+. Group C synaptotagmins are characterized by weak Ca2+-dependent (Syts M) or no homo-oligomerization activity (Syt IV). Syt VII (Group D) has unique Ca2+-dependent homooligomerization properties with EC,, values of about 150 mu M Ca2+ [Fukuda, M,, and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185]. Syts IV,VIII, and XI did not show any apparent hetero-oligomerization activity, but some sets of synaptotagmin isoforms can hetero-oligomerize in a Ca2+-dependent and/or -independent manner. Our data suggest that Ca2+-dependent and -independent hetero-oligomerization of synaptotagmins may create a variety of Ca2+-sensors.