In vitro investigation of trans SNARE complexes arrested between artificial membranes
von Halenur Yavuz
Datum der mündl. Prüfung:2014-11-21
Betreuer:Prof. Dr. Reinhard Jahn
Gutachter:Prof. Dr. Reinhard Jahn
Gutachter:Prof. Dr. Dirk Görlich
EnglischThe key components of the machinery that catalyzes membrane fusion include membrane-associated small proteins that are termed as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Calcium-triggered synaptic vesicle exocytosis is one of the most investigated SNARE-mediated fusion processes. Three synaptic SNARE proteins zipper into ternary complexes from their membrane-distant N-termini towards their membrane proximal C-termini. This assembly drives the merger of the synaptic vesicles with the pre-synaptic membrane. After the exocytosis, ternary complexes are disassembled into reactivated monomers for subsequent rounds of neurotransmitter release. This cycle includes a transient assembly intermediate that forms as the synaptic vesicle SNARE (synaptobrevin) starts zippering with the pre-synaptic plasma membrane SNAREs (syntaxin and SNAP-25). Prior to membrane fusion, partially zippered SNARE complexes are found in trans configurations with the transmembrane domains of synaptobrevin and syntaxin residing in two opposing membranes. These complexes are the substrates of various regulatory factors that control the calcium-triggered release process tightly. Current models of this regulation are based on the indirect measurements of membrane fusion probes. It was not possible so far to address the protein-protein interactions directly due to difficulties in isolating the short-lived trans SNARE complexes. This study presents a biochemically well-defined reconstitution system that captures such trans complexes successfully. SNARE zippering was artificially arrested between docking, but not-fusing large liposomes (diameter, 100 nm) by using mutants of synaptobrevin. The disassembly machinery (NSF and α-SNAP) and tetanus neurotoxin were effectively incorporated into this system. Several biochemical assays were developed utilizing these factors to study the characteristics of the trans complexes. The assembly and disassembly cycles of the trans SNARE complexes were monitored directly via fluorescently labeled proteins or indirectly via fluorescently labeled lipids. These measurements were extremely helpful in deducing the extent of SNARE zippering. Each of the two synaptobrevin mutants was involved in very different trans configurations depending on the region where their zippering was arrested, one being loosely and the other being tightly zippered. These advances proved this reconstitution system as an ideal medium for further investigations. It is made possible to address the key steps that regulate the progression from loose to tight zippering directly. Understanding this regulation is crucial for elucidating the step-wise mechanisms that attain high accuracy and speed in synaptic transmission.
Keywords: SNARE; membrane fusion; neuronal exocytosis