A REVISED MODEL FOR THE OLIGOMERIC STATE OF THE N-ETHYLMALEIMIDE-SENSITIVE FUSION PROTEIN, NSF

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking, Previou s reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for effici ent activity in membrane transport reactions. However, in recent elect ron microscopic analyses NSF appears as a hexagonal cylinder similar i n size to related ATPases known to be hexamers. We have therefore reev aluated NSF's oligomeric state using a variety of quantitative biophys ical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibri um results further suggest that the NSF hexamer is held together by ol igomerization of its D2 domains. The sedimentation coefficient, s(20,w )(0), of 13.4 (+/-0.1) S indicates that NSF has unusual hydrodynamic c haracteristics that cannot be solely explained by its shape. The demon stration that NSF is a hexameric oligomer highlights structural simila rities between it and several related ATPases which act by switching t he conformational states of their protein substrates in order to activ ate them for subsequent reactions.