Calcium sensitive ring-like oligomers formed by synaptotagmin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 111; no. 38; pp. 13966 - 13971
• Main Authors: Wang, Jing, Bello, Oscar, Auclair, Sarah M., Coleman, Jeff, Pincet, Frederic, Krishnakumar, Shyam S., Sindelar, Charles V., Rothman, James E.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 23.09.2014; National Acad Sciences
• Subjects: Biological Sciences; Calcium; Calcium - chemistry; Cell membranes; electron microscopy; Exocytosis; Humans; Lipid Bilayers - chemistry; Lipids; Magnesium; Membranes; Molecules; Multiprotein Complexes - chemistry; Multiprotein Complexes - ultrastructure; Neuroscience; Neurotransmitters; Oligomers; P branes; Physiological regulation; plasma membrane; Protein Structure, Tertiary; Proteins; SNARE Proteins - chemistry; synaptic transmission; Synaptotagmin I - chemistry; Synaptotagmins
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1415849111

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT’s cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18–43 nm, corresponding to 11–26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N -ethylmaleimide–sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. Significance Synaptotagmin-1 is the calcium sensor for synchronous neurotransmitter release. It couples calcium influx to the soluble N -ethylmaleimide–sensitive factor activating protein receptor (SNARE)-catalyzed fusion, but how this coupling happens is unknown. Here, using electron microscopy, we report that the cytosolic domain of synaptotagmin can assemble into ring-like oligomers under calcium-free conditions, and these rings disassemble rapidly upon calcium binding. This process suggests a novel but speculative mechanism to explain calcium coupling, in which the synaptotagmin rings separate the vesicle and plasma membranes and prevent the completion of SNARE complex assembly until the influx of calcium.