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

Abstract

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate1,2, characterized by a ‘stalk’ in which only the inner monolayers of the two compartments have merged to form a localized non-bilayer connection1-3. 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 catalyst4-6, is directly coupled to membrane remodeling. We used intra-molecular chemical cross-linking to stabilize dynamin in its GDP•AlF4--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 reaction7-8, the force bimodality might constitute a general paradigm for leakage-free membrane remodeling.

Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily

“…Consecutive rounds of GTP hydrolysis could sequentially increase membrane curvature. As constriction by the dynamin coat proceeds, the inner leaflets of the membrane are thought to fuse at a critical tubule diameter, achieving a hemifission intermediate . Destabilization of the membrane seems to be the strongest at the edge of an assembled and constricted dynamin helical filament, where the change in membrane curvature is highest .…”

“…Destabilization of the membrane seems to be the strongest at the edge of an assembled and constricted dynamin helical filament, where the change in membrane curvature is highest . Completion of membrane fission may be promoted by membrane insertion of hydrophobic residues from the PH domain and the dissociation of dynamin oligomers following GTP hydrolysis . Such a fission mechanism may also apply to the close homologues DNM1L and MxA, although the membrane‐dependent function of the latter is unclear.…”

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State

“…Membrane fusion is vital for eukaryotic live, in this context it has recently been shown the transition to full membrane fusion can be determined by competition between fusion and DNM2-dependent fission mechanisms supporting the hemi-fusion and hemi-fission hypothesis in live cells (Zhao et al., 2016). Our data suggest that DNM2 might play a multifaceted role during HIV-1 entry: first, a low DNM2 oligomeric state (n = 4) might help to induce HIV-1 hemi-fusion (Montessuit et al., 2010) and in turn prevent fission from happening as DNM2 fission depends on the formation of an octamer with a ring like structure and GTPase activity (Mattila et al., 2015). These sequences of events would favor HIV-1 full fusion and second, DNM2 tetramers could concomitantly stabilize the fusion pore right after HIV-1 hemi-fusion (Figure 6).…”

Identification and function of conformational dynamics in the multidomain GTP ase dynamin

“…Upon recruitment of dynamin to membranes, the PHD stably inserts its hydrophobic variable loops, VL1 and VL3, into the outer leaflet of the lipid bilayer. In combination with dynamin's assembly into a helical scaffold, wedging and tilting of the PHD drive curvature generation (Ramachandran & Schmid, ; Ramachandran et al , ) and have been proposed to be necessary for hemi‐fission and subsequent fission (Shnyrova et al , ; Sundborger et al , ; Mattila et al , ). Recent evidence suggests that the PHD wedging activity may be coupled to GTP hydrolysis (Mehrotra et al , ) and increases when the BSE is locked in the transition state (Mattila et al , ).…”