ZFIN Image: Gao et al., 2023, Figure 6.

Image

Figure Caption

Figure 6. I209N NSF-αSNAP form complexes with either ternary or binary neuronal SNARE complexes

(A) Cryo-EM structure of NSF 20S complex (PDB: 6MDM).

(B) N-D1 linker region. I209 is shown as a red stick model.

(C) Size-exclusion chromatography (SEC) profile of t20S consisting of I209N NSF, ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) SNARE complex (tSNARE), and αSNAP mixed in 1:5:25 M ratios. Predicted molecular weights for the SEC are indicated above the dotted line.

(D) SEC of b20S consisting of I209N NSF, binary (syntaxin-1A-SNAP-25) SNARE complex (bSNARE), and αSNAP mixed in 1:5:25 M ratios.

(E) SDS-PAGE gel of fractions from SEC of t20S. The elution volumes are indicated above the gel.

(F) SDS-PAGE of protein ladder and gel fractions from SEC of b20S.

(G) SDS-PAGE gel of protein ladder, NSF stock, tSNARE, bSNARE, αSNAP, t20S filtered through a 100k MWCO concentrator, and b20S filtered through a 100k MWCO concentrator.

SEC peaks are typical of a 20S complex preparation with WT NSF.⁵ The sizes of the protein species are as follows: I209N NSF, 82.8 kDa monomeric and 496.8 kDa hexameric; αSNAP, 33.3 kDa; ternary SNARE complex, 65.2 kDa; binary SNARE complex, 54.4 kDa.

Acknowledgments

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