Abstract
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.
Highlights
Mechanisms of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission are poorly understood
Outer (o) N- and C-terminal borders of variable domain (VD) deletions corresponded to transitions of ␣-helices of the stalk domain to disordered sequences that link to the PH domain in dynamin 1
The PH domain of dynamin has an essential role in targeting the endocytosis motor to the phosphoinositide-rich plasma membrane; in addition, insertion of the PH domain has been postulated to perturb the outer leaflet of the lipid bilayer to complete membrane scission [9, 10]
Summary
Mechanisms of Drp1-mediated mitochondrial fission are poorly understood. Results: Substitution of the Drp variable domain causes a spectrum of assembly and activity phenotypes but does not compromise the stalk domain-mediated recruitment of Drp to the mitochondrial anchoring protein Mff. 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 Drp assembly rate and/or geometry This in turn modulates Arg-376-dependent OMM targeting of Drp via multivalent interactions with Mff. Fission and fusion reactions determine mitochondrial shape and interconnectivity and are critical for biogenesis as well as recycling of the organelle by mitophagy. At the border of the VD and GED is a PKA phosphorylation site that inhibits mitochondrial fission [16, 17] This highly conserved serine is at positions 656 and 637 in the longest Drp splice forms of rat and human, respectively but is referred to here as SerPKA for simplicity. We provide evidence that SerPKA phosphorylation inhibits mitochondrial fission by disrupting Drp assembly and identify a residue in the Drp stalk domain necessary for Mff-dependent recruitment to mitochondria
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