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 Ca(2+)-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 Ca(2+) [Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427]. Group B Syts (I, II, VIII, and XI) show moderate homo-oligomerization irrespective of the presence of Ca(2+). Group C synaptotagmins are characterized by weak Ca(2+)-dependent (Syts IX) or no homo-oligomerization activity (Syt IV). Syt VII (Group D) has unique Ca(2+)-dependent homo-oligomerization properties with EC(50) values of about 150 microM Ca(2+) [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 Ca(2+)-dependent and/or -independent manner. Our data suggest that Ca(2+)-dependent and -independent hetero-oligomerization of synaptotagmins may create a variety of Ca(2+)-sensors.
Fukuda
M
M
Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
Mikoshiba
K
K
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050857:Synaptotagmins; D014162:Transfection
D002135:Calcium-Binding Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D002118:Calcium