Fluid cardiolipin (CL) promotes self-assembly of Drp1, a dynamin-family GTPase involved in mitochondrial fission. Drp1 sequesters CL into condensed membrane platforms and in a GTP-dependent manner increases the propensity of the lipid to undergo a nonbilayer phase transition. CL reorganization generates local membrane constriction for fission.

Background: Drp1 oligomerization and activity is critical for mitochondrial fission. Results: GTP hydrolysis is required for Drp1 constriction of lipid bilayers. The variable domain of Drp1 regulates self-assembly and is not required for constriction of lipid bilayers. Conclusion:The core machinery of Drp1 is sufficient to mediate lipid assembly, constriction, and disassembly. Significance: Characterization of the mechanoenzymatic properties of Drp1 advances our understanding of mitochondrial fission.

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 ⌬VD 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.Mitochondria undergo continuous cycles of fission and fusion to maintain a functional organelle network within eukaryotic cells. This mitochondrial network is crucial for ATP generation, apoptotic signaling, and calcium homeostasis. When the proper balance of mitochondrial dynamics is disrupted, mitochondrial dysfunction is observed (1, 2). This insult is associated with increased cell death in several human diseases, including neurodegenerative disorders (3, 4), ischemiareperfusion injury (5, 6), and glaucoma (7). Therefore, mitochondrial division has developed into a compelling therapeutic target for intervention with small molecule and peptide inhibitors that limit cell death in several of these pathologies (8 -13).The master regulator of mitochondrial fission, dynamin-related protein 1 (Drp1), 2 has been targeted in these diseases. Similar to other dynamin family members, Drp1 is a large GTPase that mediates membrane remodeling. The primary sequence of Drp1 is composed of four conserved regions (see Fig. 1A): the GTPase domain, middle domain, variable domain (VD), and GTPase effector domain (GED). Hydrolysis of GTP triggers conformational changes in Drp1 oligomers that generate the mechanical force needed to promote mitochondrial membrane scission (14, 15), and factors that inhibit Drp1 GTPase activity prevent mitochondrial division (8,16,17). The middle and GED domains promote Drp1 self-assembly, which is also critical for its role in facilitating...

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.