By contrast, the relative populations of dimeric and tetrameric species were different for each mutant as were their peak shapes. self-assembly Rabbit Polyclonal to ZADH2 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. Keywords:Human Genetics, Mitochondria, Neurodegeneration, Neurological Diseases, Protein Assembly, GTPase, Fission == Introduction == Mitochondria are critical organelles that generate ATP for cellular energy consumption. In addition, they are involved in redox and metabolic regulation, maintenance of calcium homeostasis, signaling, and fatty acid oxidation. Mitochondria undergo frequent fusion and fission events depending on cell type that are responsible for proper mitochondrial function as well as maintaining mitochondrial size, shape, and cellular distribution. Alterations in the balance of mitochondrial fusion and fission have been implicated in physiologic mechanisms such as cell division, chemotaxis, and neuronal dendrite development as well as in pathogenic processes such as apoptosis, autophagy, aging, and neurodegeneration (112). Several GTPases in the dynamin superfamily, including the mitofusins Mfn1/Mfn2, OPA1, and dynamin-related protein 1 (Drp1),6are responsible for fusion and fission of mitochondria (1315). Pathologic mutations in a number of these proteins cause autosomal dominant neurological disorders such as Charcot-Marie-Tooth neuropathy type 2A (Mfn2) and OPA1 (4), underscoring the importance of maintenance of mitochondrial morphology in mechanisms of neurodegeneration. Drp1 is an evolutionally conserved, multimeric GTPase required for mitochondrial fission. Interestingly, it also has been implicated in peroxisomal division (20). Drp1 likely mediates mitochondrial and peroxisomal fission through the formation of large multimeric spirals at mitochondrial fission sites, similar to those formed by dynamin at sites of endocytosis (1619). Similar to dynamin, Drp1 is a multidomain GTPase that consists of a GTPase domain, a middle assembly domain, a B domain of unknown function, and a GTPase-effector domain (GED). However, Drp1 lacks the pleckstrin homology domain and OAC2 C-terminal proline-rich domain found in dynamin (21). Intermolecular interactions among Drp1 monomers and intramolecular interactions between the N-terminal OAC2 GTP binding domain and the C-terminal GED are critical for Drp1 assembly and functional regulation (2225). The dynamin middle domain is critical for dynamin tetramer formation as well as higher order assembly on membranes (26). Similarly, mutations within the middle domain of Dnm1, the Drp1 ortholog in the budding yeastSaccharomyces cerevisiae, disrupt the formation of mitochondrial fission complexes (27,28). In mammalian cells, peroxisomal and mitochondrial division are impaired by a Drp1 middle domain mutation at conserved residue 363 (G363D) on one OAC2 allele (29). Together, these results suggest that middle domain-dependent assembly of Drp1 may have an important role in regulating both Drp1 function and mitochondrial morphology. Several years ago, ade novomutation in one allele at conserved residue 395 (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia; she died at 37 days (30). Mitochondria within cultured skin fibroblasts derived from this patient were markedly elongated, but the molecular mechanisms underlying these findings were not investigated. Given the importance of the middle domain in dynamin self-assembly and activity, we hypothesized that the Drp1 A395D patient mutation may be preventing Drp1 higher order assembly, leading to defects in mitochondrial fission. We, therefore, examined the effects of this A395D mutation and two other mutations at highly conserved middle domain residues (G350D, G363D) on Drp1 self-assembly OAC2 and function. Our results indicate that the Drp1 A395D lethal defect and these two other middle domain mutations impair higher order assembly of Drp1 at mitochondria, leading to markedly elongated mitochondria, presumably from decreased fission. == EXPERIMENTAL PROCEDURES == == == == == == DNA Constructs == Eukaryotic expression vectors for Myc- and HA epitope-tagged pGW1-Drp1 have been described previously (24). For yeast two-hybrid assays, wild-type and mutated Drp1 fragments were cloned into pGAD10 (prey) and pBHA (bait) vectors as described previously (24,25). Drp1 middle domain fragments (residues 332502) were generated by PCR usingPfuTurbo DNA polymerase (Agilent Stratagene, La Jolla, CA) as EcoRI/XhoI fragments and were subcloned into pGAD10/GADT7 and pBHA. The K38A, A395D, G350D, and G363D mutations were introduced into all constructs using the QuikChange method (Agilent Stratagene). ==.
