Hughson, Frederick McLauryPeng, Adele Rui-Yang2025-08-052025-08-052025-04-14https://theses-dissertations.princeton.edu/handle/88435/dsp01k643b462fIntrinsically disordered soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are the conserved machinery that facilitate membrane fusion in eukaryotic cells. The assembly of SNAREs into folded four-SNARE bundles bridging two membranes to be fused provides the driving force for fusion. Two families of protein complexes, multisubunit tethering complexes (MTC) and Sec1/Munc18 (SM) proteins, both regulate the complementarity of the SNARE combinations that are formed to ensure that fusion on occurs on-pathway, and to accelerate the rate by which SNARE assembly and fusion occur. After fusion occurs, SNARE complexes are pried apart by the conserved NSF (N-ethylmaleimide-sensitive factor) and αSNAP (soluble NSF attachment protein) proteins and individual SNAREs are recycled. Rather unexpectedly, biochemical studies implicate NSF and αSNAP in catalyzing pre-fusion SNARE assembly and accelerating fusion through synergistic action with MTCs and SM proteins. Through a series of size-exclusion chromatography (SEC)-based binding assays, we establish that the Saccharomyces cerevisiae NSF and αSNAP homologs Sec18 and Sec17 cooperatively and stably associate with Dsl1 and Sly1—the MTC and SM of the retrograde cis-Golgi/endoplasmic reticulum (ER), respectively— in a SNARE-dependent manner. Coupled with preliminary cryoelectron microscopy (cryoEM) analysis of the Dsl1 • SNARE complex • Sec17/Sec18 complex, the data suggests that the MTC and Sec17/Sec18 may not interact directly at all, with the MTC acting at N-terminus of the SNARE complex and Sec17/Sec18 at the opposite, C-terminal end. This is the first-ever study aimed at determining the structure of a pre-fusion NSF/αSNAP complex.en-USPrinceton University Senior Theses