Journal Article

Prevalent mechanism of membrane bridging by synaptotagmin-1

Liang Shi, Josep Rizo, Qiu-Xing Jiang, Alpay B. Seven, Kyle D. Brewer

2013

Overview

Synaptotagmin-1 functions as a Ca ²⁺ sensor in neurotransmitter release through its two C ₂ domains (the C ₂A and C ₂B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble N-ethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C ₂AB) was shown to bind simultaneously to two membranes via the Ca ²⁺-binding loops at the top of both domains and basic residues at the bottom of the C ₂B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC ₂AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC ₂AB oligomers bound to each membrane via the Ca ²⁺-binding loops, with no contact of the bottom of the C ₂B domain with the membranes. We now show that lnC ₂AB containing impurities indeed aggregates in solution, but properly purified lnC ₂AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC ₂AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C ₂B domain contacts the membrane in a sizeable population of molecules of both membrane-bound C ₂AB and membrane-bound lnC ₂AB. NMR data on nanodiscs show that a fraction of C ₂AB molecules bind to membranes with antiparallel orientations of the C ₂ domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C ₂AB and lnC ₂AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Biological Sciences / Calcium / Carbon Radioisotopes / Chromatography, Gel / Chromatography, Ion Exchange / Cryoelectron Microscopy / Escherichia coli / Fluorescence / fluorescence emission spectroscopy / Magnetic Resonance Spectroscopy / membrane fusion / Membranes / Models, Molecular / Molecules / Mutagenesis, Site-Directed / Neurotransmitters / nuclear magnetic resonance spectroscopy / PNAS Plus / Proteins / receptors / Spectrophotometry / Spin Labels / synaptic transmission / Synaptotagmin I - chemistry / Tritium