Abstract
The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical 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 equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.
Highlights
It is apparent that inactivation of N-ethylmaleimide-sensitive fusion protein (NSF), either by treatment with N-ethylmaleimide or by mutation of residues critical for ATP binding and hydrolysis, leads to accumulation of SNARE complexes and a block in membrane transport reactions [10, 11]
These data, as well as additional data collected at different initial loading concentrations and speeds, were poorly described by a model for a single homogeneous species (Equation 1), and yielded average molecular weights of 483,000 – 492,000, considerably higher than the 255,000 expected for the previously reported trimeric NSF oligomer [12]
Similar results were independently obtained by sedimentation equilibrium analytical ultracentrifugation with the NSF preparations used for the transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analyses shown below
Summary
It is apparent that inactivation of NSF, either by treatment with N-ethylmaleimide or by mutation of residues critical for ATP binding and hydrolysis, leads to accumulation of SNARE complexes and a block in membrane transport reactions [10, 11]. Equilibrium sedimentation data of NSF samples were initially analyzed for average molecular weights in terms of a single, homogeneous species according to cr ϭ cm exp͑ Ϫ mϩ base (Eq 1)
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