A hemi-fission intermediate links two mechanistically distinct stages of membrane fission

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate(1,2), characterized by a ‘stalk’ in which only the inner monolayers of the two compartments have merged to form a localized non-bilayer connection(1-3). Formation of the hemi-fission intermediate requires energy input from proteins catalyzing membrane remodeling; however the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analyzed how the GTPase cycle of dynamin, the prototypical membrane fission catalyst(4-6), is directly coupled to membrane remodeling. We used intra-molecular chemical cross-linking to stabilize dynamin in its GDP•AlF(4)(-)-bound transition-state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fueled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction(7-8), the force bimodality might constitute a general paradigm for leakage-free membrane remodeling.