A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Ammon E Posey,Craig Blackstone,R Blake Hill,Cara Marie Manlandro,Damien Arnoult,Julia Stadler,Chuang-Rung Chang

doi:10.1074/jbc.m110.142430

Abstract

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736-1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.

Highlights

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution
Because the middle domain is important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for dynamin-related protein 1 (Drp1) tetramerization, higher order assembly, and function
In this study we have explored the molecular mechanisms of middle domain-dependent assembly of the dynamin-related GTPase Drp1 and its importance for mitochondrial fission

Summary

EXPERIMENTAL PROCEDURES

DNA Constructs—Eukaryotic expression vectors for Mycand HA epitope-tagged pGW1-Drp have been described previously [24]. Cells were harvested by centrifugation using a Sorvall GS3 rotor at 4000 rpm for 30 min and were resuspended in calmodulin-binding peptide (CBP) column buffer A (20 mM Tris, 250 mM NaCl, 2 mM CaCl2, 1 mM imidazole, pH 8.0) containing protease inhibitors (Complete, EDTA-free Protease Inhibitor Mixture; Roche Applied Science, Indianapolis, IN). GTPase assays were started by the addition of 2.5 ␮M Drp or Drp mutants in CBP-column buffer A, where the NaCl concentration had been adjusted to 500 mM to favor Drp unassembled conditions in the stock solution. For examination of the kinetic lag associated with assembly-dependent increases in GTP hydrolysis, the activities of wild-type CBP-Drp or the CBP-Drp mutants were assayed as above at 50 mM NaCl. Immunostaining and Confocal Microscopy—HeLa cells were grown on glass coverslips and transfected with wild-type or mutant Myc-Drp using Lipofectamine (Invitrogen, Carlsbad, CA). HeLa cells by sucrose density gradient centrifugation and subjected to SDS-PAGE and immunoblotting as described previously [2, 36]

Light Scattering and Refractive

Kinetic lag min

Findings

DISCUSSION