title: Allosteric modulation of Drp1 mechanoenzyme assembly and mitochondrial fission by the variable domain.
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.
authors.affiliation: Department of Pharmacology, The University of Iowa, Iowa City, Iowa 52242, USA. stefanstrack@uiowa.edu
authors.forename: Stefan
authors.initials: S
authors.lastname: Strack
authors.forename: J Thomas
authors.initials: JT
authors.lastname: Cribbs
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D034281:Dynamins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats
chemical_list: D008565:Membrane Proteins; C533267:Mff protein, human; D024101:Mitochondrial Proteins; C519851:Dnm1l protein, rat; D034281:Dynamins