Evaluation of the heterogeneous reactivity of the syntaxin molecules on the inner leaflet of the plasma membrane.
The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) protein syntaxin 1A forms nano-sized clusters (membrane rafts) on the plasma membrane (PM) that are in equilibrium with freely diffusing syntaxin molecules. SNARE-complex formation between syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa) on the PM and synaptobrevin 2 on the vesicles (trans-SNAREs) is crucial for vesicle priming and fusion. This process might be impeded by the spontaneous accumulation of non-fusogenic cis-SNARE complexes formed when all three SNARE proteins reside on the PM. We investigated the kinetics of cis-SNARE complex assembly and disassembly and both exhibited biphasic behavior. The experimental measurements were analyzed through integration of differential rate equations pertinent to the reaction mechanism and through the application of a heuristic search for time constants and concentrations using a genetic algorithm. Reconstruction of the measurements necessitated the partitioning of syntaxin into two phases that might represent the syntaxin clusters and free syntaxin outside the clusters. The analysis suggests that most of the syntaxin in the clusters is concentrated in a nonreactive form. Consequently, cis-SNARE complex assembly in the clusters is substantially slower than outside the rafts. Interestingly, the clusters also mediate efficient disassembly of cis-SNARE complexes possibly attributable to the high local concentration of complexes in the clusters area that allows efficient disassembly by the enzymatic reaction of NSF.
Department of Neurobiochemistry, Laser Laboratory for Fast Reactions in Biology, George S Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Dana
D
Bar-On
Menachem
M
Gutman
Aviv
A
Mezer
Uri
U
Ashery
Thorsten
T
Lang
Esther
E
Nachliel
D000818:Animals; D004734:Energy Metabolism / Q000235:genetics / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D021962:Membrane Microdomains / Q000378:metabolism* / Q000502:physiology; D005810:Multigene Family / Q000502:physiology; D016716:PC12 Cells; D011485:Protein Binding / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism
D050765:Qa-SNARE Proteins; D050600:SNARE Proteins