Abstract
N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.
Highlights
Transport within the eukaryotic cell distributes cargo molecules to the proper membrane-bound compartments and maintains the distinct protein and lipid composition of organelles
Our results demonstrate that the oligomerization of both N-Ethylmaleimide-sensitive factor (NSF) N domains and ␣soluble NSF attachment proteins (SNAPs) provides a critical driving force for their interaction and the assembly of 20 S particles
Our results indicate that three ␣SNAP monomers are present in a 20 S particle, and we hypothesize that this oligomerization of ␣SNAP by SNARE complexes is sufficient to recruit a single NSF hexamer into a 20 S particle
Summary
Transport within the eukaryotic cell distributes cargo molecules to the proper membrane-bound compartments and maintains the distinct protein and lipid composition of organelles. Binding studies with these fusion proteins demonstrated that oligomerization of the N domain of NSF and trimerization of ␣SNAP are a driving force behind their interaction.
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