Binding of the Munc13-1 MUN domain to membrane-anchored SNARE complexes.
The core of the membrane fusion machinery that governs neurotransmitter release includes the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin, which form a tight "SNARE complex", and Munc18-1, which binds to the SNARE complex and to syntaxin-1 folded into a closed conformation. Release is also controlled by specialized proteins such as complexins, which also bind to the SNARE complex, and unc13/Munc13s, which are crucial for synaptic vesicle priming and were proposed to open syntaxin-1, promoting SNARE complex assembly. However, the biochemical basis for unc13/Munc13 function and its relationship to other SNARE interactions are unclear. To address this question, we have analyzed interactions of the MUN domain of Munc13-1, which is key for this priming function, using solution binding assays and cofloatation experiments with SNARE-containing proteoliposomes. Our results indicate that the Munc13-1 MUN domain binds to membrane-anchored SNARE complexes, even though binding is barely detectable in solution. The MUN domain appears to compete with Munc18-1 but not with complexin-1 for SNARE complex binding, although more quantitative assays will be required to verify these conclusions. Moreover, our data also uncover interactions of membrane-anchored syntaxin-1/SNAP-25 heterodimers with the MUN domain, Munc18-1 and complexin-1. The interaction with complexin-1 is surprising, as it was not observed in previous solution studies. Our results emphasize the importance of studying interactions within the neurotransmitter release machinery in a native membrane environment, and suggest that unc13/Munc13s may provide a template to assemble syntaxin-1/SNAP-25 heterodimers, leading to an acceptor complex for synaptobrevin.
Guan
Rong
R
Department of Biochemistry, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Dai
Han
H
Rizo
Josep
J
D000818:Animals; D002850:Chromatography, Gel; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C103912:Unc13a protein, rat