Mitochondrial Fusion Factors Research Articles

Acquired Expression of Mutant Mitofusin 2 Causes Progressive Neurodegeneration and Abnormal Behavior.

Neurons have high plasticity in developmental and juvenile stages that decreases in adulthood. Mitochondrial dynamics are highly important in neurons to maintain normal function. To compare dependency on mitochondrial dynamics in juvenile and adult stages, we generated a mouse model capable of selective timing of the expression of a mutant of the mitochondrial fusion factor Mitofusin 2 (MFN2). Mutant expression in the juvenile stage had lethal effects. Contrastingly, abnormalities did not manifest until 150 d after mutant expression during adulthood. After this silent 150 d period, progressive neurodegeneration, abnormal behaviors, and learning and memory deficits similar to those seen in human neurodegenerative diseases were observed. This indicates that abnormal neuronal mitochondrial dynamics seriously affect survival during early life stages and can also significantly damage brain function after maturation. Our findings highlight the need to consider the timing of disease onset in mimicking human neurodegenerative diseases.SIGNIFICANCE STATEMENT To compare the dependency on mitochondrial dynamics in neurons in juvenile and adult stages, we generated a mouse model expressing a mutant of the mitochondrial fusion factor MFN2 in an arbitrary timing. Juvenile expression of the mutant showed acute and severe phenotypes and had lethal effects; however, post-adult expression induced delayed but progressive phenotypes resembling those found in human neurodegenerative diseases. Our results indicate that abnormal neuronal mitochondrial dynamics seriously affect survival during early life stages and can also significantly damage brain function after maturation. This strongly suggests that the timing of expression should be considered when establishing an animal model that closely resembles human neurodegenerative diseases.

Betaine enhances the cellular survival via mitochondrial fusion and fission factors, MFN2 and DRP1

ABSTRACTBetaine is a key metabolite of the methionine cycle and known for attenuating alcoholic steatosis in the liver. Recent studies have focused on the protection effect of betaine in mitochondrial regulation through the enhanced oxidative phosphorylation system. However, the mechanisms of its beneficial effects have not been clearly identified yet. Mitochondrial dynamics is important for the maintenance of functional mitochondria and cell homeostasis. A defective mitochondrial dynamics and oxidative phosphorylation system have been closely linked to several pathologies, raising the possibility that novel drugs targeting mitochondrial dynamics may present a therapeutic potential to restore the cellular homeostasis. In this study, we investigated betaine’s effect on mitochondrial morphology and physiology and demonstrated that betaine enhances mitochondrial function by increasing mitochondrial fusion and improves cell survival. Furthermore, it rescued the unbalance of the mitochondrial dynamics from mitochondrial oxidative phosphorylation dysfunction induced by oligomycin and rotenone. The elongation properties by betaine were accompanied by lowering DRP1 and increasing MFN2 expression. These data suggest that betaine could play an important role in remodeling mitochondrial dynamics to enhance mitochondrial function and cell viability.

Tributyltin Inhibits Neural Induction of Human Induced Pluripotent Stem Cells

Tributyltin (TBT), one of the organotin compounds, is a well-known environmental pollutant. In our recent study, we reported that TBT induces mitochondrial dysfunction, in human-induced pluripotent stem cells (iPSCs) through the degradation of mitofusin1 (Mfn1), which is a mitochondrial fusion factor. However, the effect of TBT toxicity on the developmental process of iPSCs was not clear. The present study examined the effect of TBT on the differentiation of iPSCs into the ectodermal, mesodermal, and endodermal germ layers. We found that exposure to nanomolar concentration of TBT (50 nM) selectively inhibited the induction of iPSCs into the ectoderm, which is the first step in neurogenesis. We further assessed the effect of TBT on neural differentiation and found that it reduced the expression of several neural differentiation marker genes, which were also downregulated by Mfn1 knockdown in iPSCs. Taken together, these results indicate that TBT induces developmental neurotoxicity via Mfn1-mediated mitochondrial dysfunction in iPSCs.

Novel role of mitochondrial fusion factor Mfn1 in neural differentiation of human iPS cells

Human induced pluripotent stem cells (iPSCs) are expected as potential sources that provide human neural cells for drug development. However, the mechanisms underlying the neural differentiation of iPSCs remain largely unknown.

Prevention of recognition memory loss and moderation of mitochondrial dynamic tendency toward fusion by flavone derivatives in Aβ-injected rats: a comparison between two flavonoids with different polarity

Growing evidence sheds light on the use of flavonoids as the promising alternatives for the treatment of chronic conditions, including cancer and neurodegenerative disorders. Accordingly, in the present study, we aimed at evaluating the effects of oral intake of two structurally different flavonoids 5-hydroxy-6,7,4ʹ-trimethoxyflavone (flavone 1) and 5,7,4ʹ-trihydroxyflavone (flavone 2) on recognition memory, hippocampal protein level of immediate early gene cFos and mitochondrial dynamic markers in Amyloid β (Aβ)-injected rats. Recognition aspect of memory and level of proteins were measured using novel object recognition test and Western blot, respectively. Our data indicated that even though flavone 1 was more effective than flavone 2 to prevent memory impairment, feeding with both flavones alleviated memory in Aβ-injected rats. Furthermore, in flavones-administered rats, mitochondrial dynamic balancing returned to the control level by the decline in Dynamin-related protein-1 protein level, a known marker for mitochondrial fission, and elevation in protein level of mitochondrial fusion factors Mitofusins 1 and 2. In parallel with behavior results, flavone 1 was more effectual on mitochondrial dynamic moderating. The more neuroprotective effects of flavone 1 could be attributed to its methylated structure leading to crossing of the blood-brain barrier with ease and metabolic stability and bioactivity.

Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding

Defective RNA metabolism and transport are implicated in aging and degeneration [1, 2], but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration [3]. Here, we link a novel mechanism for RNA export through nuclear envelope (NE) budding [4, 5] that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. We identified mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin), as well as other transcripts required for mitochondrial integrity and function, in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates [6, 7] and myofibril disorganization [8, 9]. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marfwas insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis ofNE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging.

A mitochondrial division inhibitor, Mdivi-1, inhibits mitochondrial fragmentation and attenuates kainic acid-induced hippocampal cell death.

BackgroundKainic acid (KA)-induced excitotoxicity promotes cytoplasmic calcium accumulation, oxidative stress, and apoptotic signaling, leading to hippocampal neuronal death. Mitochondria play a critical role in neuroinflammation and the oxidative stress response. Mitochondrial morphology is disrupted during KA-induced seizures; however, it is not clear whether mitochondrial fission or fusion factors are involved in KA-induced neuronal death.ResultsWe investigated the effect of Mdivi-1, a chemical inhibitor of the mitochondrial fission protein Drp1, on mitochondrial morphology and function in KA-injected mice. Mdivi-1 pretreatment significantly reduced seizure activity and increased survival rates of KA-treated mice. Mdivi-1 was protective against mitochondrial morphological disruption, and it reduced levels of phosphorylated Drp1 (Ser616) and Parkin recruitment to mitochondria. By contrast, levels of mitochondrial fusion factors did not change. Mdivi-1 also reduced KA-induced neuroinflammation and glial activation.ConclusionsWe conclude that inhibition of mitochondrial fission attenuates Parkin-mediated mitochondrial degradation and protects from KA-induced hippocampal neuronal cell death.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-016-0270-y) contains supplementary material, which is available to authorized users.

Association between myocyte quality control signaling and sarcopenia in old hip-fractured patients: Results from the Sarcopenia in HIp FracTure (SHIFT) exploratory study

BackgroundSarcopenia has been proposed as a potentially amenable factor impacting the clinical outcomes of hip-fractured elderly. The identification of specific biological targets is therefore crucial to developing pharmacological interventions against age-related muscle wasting. The present work reports promising preliminary data on the association between alterations of myocyte quality control (MQC) signaling and sarcopenia in old patients with hip fracture. MethodsTwenty-five elderly hip-fractured patients (20 women and 5 men; mean age 84.9±1.65years) were enrolled as part of the Sarcopenia in HIp FracTure (SHIFT) study. Intraoperative biopsies of the vastus lateralis muscle were obtained and assayed for the expression of a set of MQC signaling proteins. The presence of sarcopenia was established according to the European Working Group on Sarcopenia in Older People (EWGSOP) criteria, with bioelectrical impedance analysis used for fat-free mass estimation. ResultsSarcopenia was identified in 10 patients (40%). Protein expression of the mitochondrial fusion factor mitofusin (Mfn) 2 and the autophagy mediator microtubule-associated protein 1 light chain 3B (LC3B) was significantly lower in patients with sarcopenia compared with non-sarcopenic controls. No differences between groups were observed for Mfn1, optic atrophy protein 1 (OPA1), fission protein 1 (Fis1), and the master regulator of mitochondrial biogenesis peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). ConclusionData from this exploratory study show that a reduced expression of the mitochondrial fusion factor Mfn2 and the autophagy mediator LC3B is associated with sarcopenia in old hip-fractured patients. Future larger-scale studies are needed to corroborate these preliminary findings and determine whether MQC pathways may be targeted to improve muscle health and promote functional recovery in old patients with hip fracture.

Mitochondrial Fission and Fusion Factors Reciprocally Orchestrate Mitophagic Culling in Mouse Hearts and Cultured Fibroblasts

How mitochondrial dynamism (fission and fusion) affects mitochondrial quality control is unclear. We uncovered distinct effects on mitophagy of inhibiting Drp1-mediated mitochondrial fission versus mitofusin-mediated mitochondrial fusion. Conditional cardiomyocyte-specific Drp1 ablation evoked mitochondrial enlargement, lethal dilated cardiomyopathy, and cardiomyocyte necrosis. Conditionally ablating cardiomyocyte mitofusins (Mfn) caused mitochondrial fragmentation with eccentric remodeling and no cardiomyocyte dropout. Parallel studies in cultured murine embryonic fibroblasts (MEFs) and in vivo mouse hearts revealed that Mfn1/Mfn2 deletion provoked accumulation of defective mitochondria exhibiting an unfolded protein response, without appropriately increasing mitophagy. Conversely, interrupting mitochondrial fission by Drp1 ablation increased mitophagy and caused a generalized loss of mitochondria. Mitochondrial permeability transition pore (MPTP) opening in Drp1 null mitochondria was associated with mitophagy in MEFs and contributed to cardiomyocyte necrosis and dilated cardiomyopathy in mice. Drp1, MPTP, and cardiomyocyte mitophagy are functionally integrated. Mitochondrial fission and fusion have opposing roles during in vivo cardiac mitochondrial quality control.

Dissociation of mitochondrial from sarcoplasmic reticular stress in Drosophila cardiomyopathy induced by molecularly distinct mitochondrial fusion defects

Mitochondrial dynamism (fusion and fission) is responsible for remodeling interconnected mitochondrial networks in some cell types. Adult cardiac myocytes lack mitochondrial networks, and their mitochondria are inherently “fragmented”. Mitochondrial fusion/fission is so infrequent in cardiomyocytes as to not be observable under normal conditions, suggesting that mitochondrial dynamism may be dispensable in this cell type. However, we previously observed that cardiomyocyte-specific genetic suppression of mitochondrial fusion factors optic atrophy 1 (Opa1) and mitofusin/MARF evokes cardiomyopathy in Drosophila hearts. We posited that fusion-mediated remodeling of mitochondria may be critical for cardiac homeostasis, although never directly observed. Alternately, we considered that inner membrane Opa1 and outer membrane mitofusin/MARF might have other as-yet poorly described roles that affect mitochondrial and cardiac function. Here we compared heart tube function in three models of mitochondrial fragmentation in Drosophila cardiomyocytes: Drp1 expression, Opa1 RNAi, and mitofusin MARF RNA1. Mitochondrial fragmentation evoked by enhanced Drp1-mediated fission did not adversely impact heart tube function. In contrast, RNAi-mediated suppression of either Opa1 or mitofusin/MARF induced cardiac dysfunction associated with mitochondrial depolarization and ROS production. Inhibiting ROS by overexpressing superoxide dismutase (SOD) or suppressing ROMO1 prevented mitochondrial and heart tube dysfunction provoked by Opa1 RNAi, but not by mitofusin/MARF RNAi. In contrast, enhancing the ability of endoplasmic/sarcoplasmic reticulum to handle stress by expressing Xbp1 rescued the cardiomyopathy of mitofusin/MARF insufficiency without improving that caused by Opa1 deficiency. We conclude that decreased mitochondrial size is not inherently detrimental to cardiomyocytes. Rather, preservation of mitochondrial function by Opa1 located on the inner mitochondrial membrane, and prevention of ER stress by mitofusin/MARF located on the outer mitochondrial membrane, are central functions of these “mitochondrial fusion proteins”.

Neural stem cell transplantation enhances mitochondrial biogenesis in a transgenic mouse model of Alzheimer's disease–like pathology

Mitochondrial dysfunction, especially a defect in mitochondrial biogenesis, is an early and prominent feature of Alzheimer's disease (AD). Previous studies demonstrated that the number of mitochondria is significantly reduced in susceptible hippocampal neurons from AD patients. Neural stem cell (NSC) transplantation in AD-like mice can compensate for the neuronal loss resulting from amyloid-beta protein deposition. The effects of NSC transplantation on mitochondrial biogenesis and cognitive function in AD-like mice, however, are poorly understood. In this study, we injected NSCs or vehicle into 12-month-old amyloid precursor protein (APP)/PS1 transgenic mice, a mouse model of AD-like pathology. The effects of NSC transplantation on cognitive function, the amount of mitochondrial DNA, the expression of mitochondrial biogenesis factors and mitochondria-related proteins, and mitochondrial morphology were investigated. Our results show that in NSC-injected APP/PS1 (Tg-NSC) mice, the cognitive function, number of mitochondria, and expression of mitochondria-related proteins, specifically the mitochondrial fission factors (dynamin-related protein 1 [Drp1] and fission 1 [Fis1]) and the mitochondrial fusion factor optic atrophy 1 (OPA1), were significantly increased compared with those in age-matched vehicle-injected APP/PS1 (Tg-Veh) mice, whereas the expression of mitochondrial fusion factors mitofusion 1 (Mfn1) and Mfn2 was significantly decreased. These data indicate that NSC transplantation may enhance mitochondria biogenesis and further rescue cognitive deficits in AD-like mice.

The mitochondrial dynamism-mitophagy-cell death interactome: multiple roles performed by members of a mitochondrial molecular ensemble.

Mitochondrial research is experiencing a renaissance, in part, because of the recognition that these endosymbiotic descendants of primordial protobacteria seem to be pursuing their own biological agendas. Not only is mitochondrial metabolism required to produce most of the biochemical energy that supports their eukaryotic hosts (us) but mitochondria can actively (through apoptosis and programmed necrosis) or passively (through reactive oxygen species toxicity) drive cellular dysfunction or demise. The cellular mitochondrial collective autoregulates its population through biogenic renewal and mitophagic culling; mitochondrial fission and fusion, 2 components of mitochondrial dynamism, are increasingly recognized as playing central roles as orchestrators of these processes. Mitochondrial dynamism is rare in striated muscle cells, so cardiac-specific genetic manipulation of mitochondrial fission and fusion factors has proven useful for revealing noncanonical functions of mitochondrial dynamics proteins. Here, we review newly described functions of mitochondrial fusion/fission proteins in cardiac mitochondrial quality control, cell death, calcium signaling, and cardiac development. A mechanistic conceptual paradigm is proposed in which cell death and selective organelle culling are not distinct processes, but are components of a unified and integrated quality control mechanism that exerts different effects when invoked to different degrees, depending on pathophysiological context. This offers a plausible explanation for seemingly paradoxical expression of mitochondrial dynamics and death factors in cardiomyocytes wherein mitochondrial morphometric remodeling does not normally occur and the ability to recover from cell suicide is severely limited.

Regulation of mitophagy by the Gp78 E3 ubiquitin ligase

Glycoprotein 78 (Gp78) is a critical E3 ubiquitin ligase in endoplasmic reticulum-associated degradation. Overexpression of Flag-tagged Gp78 (Flag-gp78), but not Flag-gp78 mutated in its RING-finger domain (Flag-RINGmut) with deficient ubiquitin ligase activity, induces mitochondrial fragmentation and ubiquitination and proteasome-dependent degradation of the mitofusin (Mfn) mitochondrial fusion factors Mfn1/Mfn2. After mitochondrial depolarization with carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flag-gp78 induced a threefold loss of depolarized mitochondria and significant loss of the inner mitochondrial protein OxPhosV. Flag-gp78-dependent loss of OxPhosV, but not Mfn1 or Mfn2, was prevented by small interfering RNA (siRNA) knockdown of the autophagy protein Atg5 in CCCP-treated cells. Gp78-induced mitophagy required ubiquitin ligase activity, as it is not observed upon transfection of Flag-RINGmut or cotransfection of Flag-gp78 with ubiquitin mutated at three critical lysine residues (K29, 48, 63R) involved in polyubiquitin chain elongation. Short hairpin RNA knockdown of Gp78 in HT-1080 fibrosarcoma cells increased mitofusin levels and reduced depolarization-induced mitophagy, whereas siRNA knockdown showed that Mfn1, but not Mfn2, was required for Gp78-dependent depolarization-induced mitophagy. Mitochondrial depolarization induced Gp78-dependent expression of the autophagic marker LC3II and recruitment of enhanced green fluorescent protein-LC3 to the Gp78- and calnexin-labeled, mitochondria-associated ER. Finally, Gp78-induced mitophagy is Parkin independent, as it occurs in Parkin-null HeLa cells and upon siRNA-mediated Parkin knockdown in HEK293 cells. This study therefore describes a novel role for the ER-associated Gp78 ubiquitin ligase and the Mfn1 mitochondrial fusion factor in mitophagy.

Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.

Mutations in PINK1 and Parkin Impair Ubiquitination of Mitofusins in Human Fibroblasts

PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy.Here, we investigated the impact of mutations in endogenous PINK1 and Parkin on the ubiquitination of mitochondrial fusion and fission factors and the mitochondrial network structure. Treating control fibroblasts with mitochondrial membrane potential (Δψ) inhibitors or H2O2 resulted in ubiquitination of Mfn1/2 but not of OPA1 or Fis1. Ubiquitination of Mitofusins through the PINK1/Parkin pathway was observed within 1 h of treatment. Upon combined inhibition of Δψ and the ubiquitin proteasome system (UPS), no ubiquitination of Mitofusins was detected. Regarding morphological changes, we observed a trend towards increased mitochondrial branching in PD patient cells upon mitochondrial stress.For the first time in PD patient-derived cells, we demonstrate that mutations in PINK1 and Parkin impair ubiquitination of Mitofusins. In the presence of UPS inhibitors, ubiquitinated Mitofusin is deubiquitinated by the UPS but not degraded, suggesting that the UPS is involved in Mitofusin degradation.

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Regulation of Mitochondrial Morphology by USP30, a Deubiquitinating Enzyme Present in the Mitochondrial Outer Membrane

Recent studies have suggested that ubiquitination of mitochondrial proteins participates in regulating mitochondrial dynamics in mammalian cells, but it is unclear whether deubiquitination is involved in this process. Here, we identify human ubiquitin-specific protease 30 (USP30) as a deubiquitinating enzyme that is embedded in the mitochondrial outer membrane. Depletion of USP30 expression by RNA interference induced elongated and interconnected mitochondria, depending on the activities of the mitochondrial fusion factors mitofusins, without changing the expression levels of the key regulators for mitochondrial dynamics. Mitochondria were rescued from this abnormal phenotype by ectopic expression of USP30 in a manner dependent on its enzymatic activity. Our findings reveal that USP30 participates in the maintenance of mitochondrial morphology, a finding that provides new insight into the cellular function of deubiquitination.

Stage-specific enhanced expression of mitochondrial fusion and fission factors during spermatogenesis in rat testis

The mitochondrial fusion factors mitofusins 1 and 2 (Mfn1 and Mfn2) and the fission factor dynamin-related protein 1 (Drp1) were found to be highly expressed in the pubertal and adult rat testis by Northern blot analysis. Immunohistochemistry using specific antisera to Mfn2 and Drp1 revealed a pronounced expression of the fusion and fission factors in the round and elongating spermatids in the seminiferous tubules, suggesting that at precise steps of spermiogenesis (i.e., steps 8–12), spermatid mitochondria are rapidly homogenized by frequent fusion and division. Although physiological relevance of this phenomenon remains to be clarified, a role is proposed for it as an effective means of achieving complete and homogeneous ubiquitination of mitochondria, which has recently been demonstrated to be a mechanism for the elimination of paternal mitochondria during fertilization, based on the fact that the timing of expression of Mfn2 and Drp1 coincides well with that reported for a spermatid-specific ubiquitin-conjugating enzyme.

Gag3p, an outer membrane protein required for fission of mitochondrial tubules.

Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane. Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth). Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures. Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide. The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission. GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay. Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane. This association requires neither the DNM1 nor GAG2 gene products. However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p. These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.