Cryo-EM Studies of Drp1 Reveal Cardiolipin Interactions that Activate the Helical Oligomer.

Dynamins are mechano-chemical GTPases involved in the remodeling of cellular membranes. In this study, we have investigated the mechanism of dynamin-related protein 1 (Drp1), a key mediator of mitochondrial fission. To date, it is unclear how Drp1 assembles on the mitochondrial outer membrane in response to different lipid signals to induce membrane fission. Here, we present cryo-EM structures of Drp1 helices on nanotubes with distinct lipid compositions to mimic membrane interactions with the fission machinery. These Drp1 polymers assemble exclusively through stalk and G-domain dimerizations, which generates an expanded helical symmetry when compared to other dynamins. Interestingly, we found the characteristic gap between Drp1 and the lipid bilayer was lost when the mitochondrial specific lipid cardiolipin was present, as Drp1 directly interacted with the membrane. Moreover, this interaction leads to a change in the helical structure, which alters G-domain interactions to enhance GTPase activity. These results demonstrate how lipid cues at the mitochondrial outer membrane (MOM) can alter Drp1 structure to activate the fission machinery.

Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity.

Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical, and cellular approaches, we find that the A- and B-inserts distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Although Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and even counter-productive at high CL concentrations, Mff stimulation of the much less cooperative longest Drp1 isoform (Drp1-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.

Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction.

Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission.

Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a cytosolic member of the dynamin superfamily of large GTPases, interacts with CL and functions to sustain the balance of mitochondrial division and fusion by catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly and GTPase activity at the membrane surface, the mechanism by which CL functions in membrane fission, if at all, remains unclear. Here, using a variety of fluorescence spectroscopic and imaging approaches together with model membranes, we demonstrate that Drp1 and CL function cooperatively in effecting membrane constriction toward fission in three distinct steps. These involve 1) the preferential association of Drp1 with CL localized at a high spatial density in the membrane bilayer, 2) the reorganization of unconstrained, fluid-phase CL molecules in concert with Drp1 self-assembly, and 3) the increased propensity of CL to transition from a lamellar, bilayer arrangement to an inverted hexagonal, nonbilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. Thus we propose that Drp1 and CL function in concert to catalyze mitochondrial division.

The mechanoenzymatic core of dynamin-related protein 1 comprises the minimal machinery required for membrane constriction.

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), a large GTPase of the dynamin superfamily, is the main mediator of mitochondrial fission. Like prototypical dynamin, Drp1 is composed of a mechanochemical core consisting of the GTPase, middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin, however, Drp1 contains an unstructured variable domain, whose function is not yet fully resolved. Here, using time-resolved EM and rigorous statistical analyses, we establish the ability of full-length Drp1 to constrict lipid bilayers through a GTP hydrolysis-dependent mechanism. We also show the variable domain limits premature Drp1 assembly in solution and promotes membrane curvature. Furthermore, the mechanochemical core of Drp1, absent of the variable domain, is sufficient to mediate GTP hydrolysis-dependent membrane constriction.