Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

46-POS Board 16 DIRECT DETECTION OF THE MOLECULAR ORGANIZATION OF VAMP2 SYNAPTOPHYSIN FROM LIPID MEMBRANE Aniruddha Panda 1,2 ; Wonhyeuk Jung 1,2 ; Jeff Coleman 1,2 ; Kallol Gupta 1,2 ; James E Rothman 1,2 ; 1 Nanobiology Institute, Yale University, West Haven, CT, USA 2 Yale School of Medicine, Department of Cell Biology, New Haven, CT, USA The molecular rationale behind the ultra-fast fusion of synaptic vesicles (SV) remains enigmatic due to our limited understanding of the supra-molecular functional organization of the involved proteins and lipids. To address this, we have developed a novel tunable lipid-vesicle native-mass spectrometry (nativeMS) platform that can determine membrane proteins-lipid complexes directly from customizable membranes. The method enables us to introduce intact lipid membranes, embedded with target membrane proteins, directly into the MS. Subsequent gentle ablation of the protein-lipid complexes from the membrane inside MS enables the determination of molecular organization states, bound lipid ID, and the binding stoichiometry of the embedded target membrane proteins at precise molecular resolution. Utilizing this platform, we discovered that VAMP2 exclusively exists as a monomer within the SV-bilayer. Through high-resolution nativeMS and top-down MS/MS analysis, we identified VAMP2's specific binding to phosphatidylcholine (PC) and cholesterol in the SV membrane. Further investigations employing fusion assays show that specific interaction between VAMP2 and PC regulates the timing of vesicle fusion. We next turn our focus on understanding the role of Synaptophysin (SYP) in templating VAMP2 in SVs. By directly studying VAMP2-Syp from the lipid membrane, we show that SYP organizes VAMP2 into higher-order complexes in an SV-like bilayer. To gain deeper insights into VAMP2's dimeric nature in the presence of synaptophysin, specific mutations were introduced to determine both the VAMP-VAMP and VAMP-SYP interfaces. Simultaneous vesicle fusion assays on these wt/mutant complexes reveal the functional role of these assemblies in regulating SV-fusion. Further expanding our platform, we screen specific VAMP2 mutations with pathophysiological effects. Together, our work presents a novel experimental platform to determine the oligomeric organization of membrane proteins and lipids directly from lipid membranes. The application of this shows how VAMP2 is organized in the membrane by synaptophysin.

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