SNARE Cis-Reassembly: Investigating SNARE Interactions Post-Disassembly

by Delane F. Espinueva
Doctoral thesis
Date of Examination:2024-06-17
Date of issue:2025-06-16
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Claudia Steinem
Persistent Address: http://dx.doi.org/10.53846/goediss-11331

 

 

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Abstract

English

Neurotransmitter release relies on the SNARE-mediated fusion of neurotransmitter-filled synaptic vesicles with the presynaptic plasma membrane. SNARE proteins from these opposing membranes interact to form a stable α-helical complex, releasing free energy that energetically drives membrane fusion. In preparation for its recycling, SNARE complexes are disassembled by the ATPase NSF, along with cofactor αSNAP. The mechanisms of SNARE disassembly and events surrounding are not well understood. Given the spontaneity of SNARE complex formation and SNARE proximity post-disassembly, it is reasonable to hypothesize that disassembled SNARE proteins will reassemble into complexes while they are on the same membrane (cis-reassembly), in the absence of regulatory factors. However, in order to reuse the SNAREs for future neurotransmitter signalling, neuronal SNARE proteins must be sorted to opposing membranes: Syntaxin 1 and SNAP-25 to the plasma membrane, and Synaptobrevin 2 to synaptic vesicles. Therefore, individual SNAREs must stay disassembled. This thesis aims to investigate how the SNAREs interact after disassembly, and whether disassembled SNAREs reassemble in the same membrane. To this end, I developed a membrane system in-vitro with minimal presence of SNARE-interacting proteins that may regulate this process. In this minimal system, I developed a method to independently membraneanchor each neuronal SNARE. Since the neuronal SNARE SNAP-25 lacks a transmembrane domain, it is difficult to anchor to membranes in-vitro. Thus, I modify a variant and show that it is able to function in SNARE assembly and disassembly, and that it is membrane-bound both before and after complex disassembly. Using this minimal system of purified proteins and liposomes, I disassemble FRET-paired SNARE complexes to observe membrane-bound SNAREs immediately after disassembly. My results show that SNARE cis-reassembly occurs but at a surprisingly slow rate. Even in a minimal system, SNARE cis-reassembly is kinetically hindered. Further investigating this process, I examined the role the disassembly machinery plays post-disassembly. My results show that αSNAP contributes to the inhibition of SNARE cis-reassembly. Finally, I discuss the implications of my main results, along with some preliminary data, and offer insights on SNARE disassembly and recycling. In addition, the minimal system developed here has applications beyond the questions addressed here.
Keywords: SNARE complex; SNARE cycle; SNARE reassembly; in-vitro lipidation
 
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