The mechanoenzyme dynamin-related protein 1 (Drp1) hydrolyzes GTP to power 
mitochondrial fission, a process required for organelle biogenesis, quality 
control, transport, and apoptosis. The pleckstrin homology domain of dynamin is 
essential for targeting to and severing of lipid tubules, but the function of 
the corresponding variable domain (VD, or insert B) of Drp1 is unknown. We 
replaced the VD of Drp1 with a panel of linker sequences of varying length and 
secondary structure composition and found that the VD is dispensable for 
mitochondrial recruitment, association with the Drp1-anchoring protein Mff 
(mitochondrial fission factor), and basal and protonophore-induced mitochondrial 
fragmentation. Indeed, several ΔVD mutants constitutively localized to the outer 
mitochondrial membrane (OMM) and fragmented mitochondria more efficiently than 
wild-type Drp1. Consistent with an autoinhibitory role of the VD, we identified 
Arg-376 in the Drp1 stalk domain as necessary for Mff interaction, assembly into 
spirals, and mitochondrial fission. Switching the length of N- and C-terminal 
α-helical segments in the VD-replacing linker converted Drp1 from constitutively 
active and OMM-localized to inactive and cytosolic. Other hypoactive ΔVD mutants 
formed stable and characteristically shaped aggregates, including extended 
filaments. Phosphorylation of a PKA site bordering the VD disassembled the 
filamentous ΔVD mutant and accelerated cytosolic diffusion of full-length Drp1. 
We propose a model for regulation of Drp1-dependent mitochondrial fission, in 
which posttranslational modifications in or near the VD alter the conformation 
of a membrane-proximal oligomerization interface to influence Drp1 assembly rate 
and/or geometry. This in turn modulates Arg-376-dependent OMM targeting of Drp1 
via multivalent interactions with Mff.