Mgm1p,a dynamin-related GTPase,is essential for fusion of the mitochondrial outer membrane

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.     

Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast

Mitochondrial fusion and fission play important roles for mitochondrial morphology and function. We identified Mdm30 as a novel component required for maintenance of fusion-competent mitochondria in yeast. The Mdm30 sequence contains an F-box motif that is commonly found in subunits of Skp1-Cdc53-F-box protein ubiquitin ligases. A fraction of Mdm30 is associated with mitochondria. Cells lacking Mdm30 contain highly aggregated or fragmented mitochondria instead of the branched tubular network seen in wild-type cells. Deltamdm30 cells lose mitochondrial DNA at elevated temperature and fail to fuse mitochondria in zygotes at all temperatures. These defects are rescued by deletion of DNM1, a gene encoding a component of the mitochondrial division machinery. The protein level of Fzo1, a key component of the mitochondrial fusion machinery, is regulated by Mdm30. Elevated Fzo1 levels in cells lacking Mdm30 or in cells overexpressing Fzo1 from a heterologous promoter induce mitochondrial aggregation in a similar manner. Our results suggest that Mdm30 controls mitochondrial shape by regulating the steady-state level of Fzo1 and point to a connection of the ubiquitin/26S proteasome system and mitochondria.     

Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins

Mitochondrial morphology depends on balanced fusion and fission events. A central component of the mitochondrial fusion apparatus is the conserved GTPase Fzo1 in the outer membrane of mitochondria. Mdm30, an F-box protein required for mitochondrial fusion in vegetatively growing cells, affects the cellular Fzo1 concentration in an unknown manner. We demonstrate that mitochondrial fusion requires a tight control of Fzo1 levels, which is ensured by Fzo1 turnover. Mdm30 binds to Fzo1 and, dependent on its F-box, mediates proteolysis of Fzo1. Unexpectedly, degradation occurs along a novel proteolytic pathway not involving ubiquitylation, Skp1-Cdc53-F-box (SCF) E3 ubiquitin ligase complexes, or 26S proteasomes, indicating a novel function of an F-box protein. This contrasts to the ubiquitin- and proteasome-dependent turnover of Fzo1 in alpha-factor-arrested yeast cells. Our results therefore reveal not only a critical role of Fzo1 degradation for mitochondrial fusion in vegetatively growing cells but also the existence of two distinct proteolytic pathways for the turnover of mitochondrial outer membrane proteins.     

Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1

Mitochondrial outer- and inner-membrane fusion events are coupled in vivo but separable and mechanistically distinct in vitro, indicating that separate fusion machines exist in each membrane. Outer-membrane fusion requires trans interactions of the dynamin-related GTPase Fzo1, GTP hydrolysis, and an intact inner-membrane proton gradient. Inner-membrane fusion also requires GTP hydrolysis but distinctly requires an inner-membrane electrical potential. The protein machinery responsible for inner-membrane fusion is unknown. Here, we show that the conserved intermembrane-space dynamin-related GTPase Mgm1 is required to tether and fuse mitochondrial inner membranes. We observe an additional role of Mgm1 in inner-membrane dynamics, specifically in the maintenance of crista structures. We present evidence that trans Mgm1 interactions on opposing inner membranes function similarly to tether and fuse inner membranes as well as maintain crista structures and propose a model for how the mitochondrial dynamins function to facilitate fusion.     

Instability of the mitofusin Fzo1 regulates mitochondrial morphology during the mating response of the yeast Saccharomyces cerevisiae

Mitochondria form a highly dynamic network that is shaped by continuous fission and fusion of these organelles. In the yeast Saccharomyces cerevisiae two machineries are involved in this process, one of which includes the mitochondrial fusion promoting GTPase Fzo1. Although a role for the F-box protein Mdm30 in regulating the stability of Fzo1 has been proposed, the molecular basis for the regulation of the fission to fusion ratio of mitochondria remains unknown. To discern the mechanism of the regulation of mitochondrial morphology, we arrested cells at different stages of the cell cycle and examined mitochondrial morphology as well as the stability of mitochondrial fission and fusion proteins. In response to a G1 arrest evoked by the mating pheromone alpha factor the mitochondrial network fragmented into small pieces, which was accompanied by dramatic down-regulation of Fzo1. Mating pheromone also triggered the degradation of Fzo1 produced under the control of a constitutive promoter, and Fzo1 was stabilized upon proteasome inhibition, indicating a role for the proteasome system in the degradation of Fzo1. However, deletion of MDM30 did not stabilize Fzo1 after mating pheromone treatment, showing a different mechanism from the previously reported process of steady state Fzo1 regulation. We show an example for a regulated change of the mitochondrial fission to fusion ratio during the life cycle of budding yeast. Proteasomal degradation of Fzo1 in response to the mating pheromone is proposed to mediate the remodeling of the mitochondrial network during the process of mating.     

Ubiquitin-proteasome-dependent degradation of a mitofusin, a critical regulator of mitochondrial fusion

The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome-dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCF(Mdm30p) ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion.     

Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion

In yeast, mitochondrial fusion requires Ugo1p and two GTPases, Fzo1p and Mgm1p. Ugo1p is anchored in the mitochondrial outer membrane with its N terminus facing the cytosol and C terminus in the intermembrane space. Fzo1p is also an outer membrane protein, whereas Mgm1p is located in the intermembrane space. Recent studies suggest that these three proteins form protein complexes that mediate mitochondrial fusion. Here, we show that the cytoplasmic domain of Ugo1p directly interacts with Fzo1p, whereas its intermembrane space domain binds to Mgm1p. We identified the Ugo1p-binding site in Fzo1p and demonstrated that Ugo1p-Fzo1p interaction is essential for the formation of mitochondrial shape, maintenance of mitochondrial DNA, and fusion of mitochondria. Although the GTPase domains of Fzo1p and Mgm1p regulate mitochondrial fusion, they were not required for association with Ugo1p. Furthermore, we found that Ugo1p bridges the interaction between Fzo1p and Mgm1p in mitochondria. Our data indicate that distinct regions of Ugo1p bind directly to Fzo1p and Mgm1p and thereby link these two GTPases during mitochondrial fusion.     

Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

UGO1 encodes an outer membrane protein required for mitochondrial fusion

Membrane fusion plays an important role in controlling the shape, number, and distribution of mitochondria. In the yeast Saccharomyces cerevisiae, the outer membrane protein Fzo1p has been shown to mediate mitochondrial fusion. Using a novel genetic screen, we have isolated new mutants defective in the fusion of their mitochondria. One of these mutants, ugo1, shows several similarities to fzo1 mutants. ugo1 cells contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. ugo1 mutants lose mitochondrial DNA (mtDNA). In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents. Fragmentation of mitochondria and loss of mtDNA in ugo1 mutants are rescued by disrupting DNM1, a gene required for mitochondrial division. We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane. Ugo1p appears to contain a single transmembrane segment, with its NH(2) terminus facing the cytosol and its COOH terminus in the intermembrane space. Our results suggest that Ugo1p is a new outer membrane component of the mitochondrial fusion machinery.     

Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion

Mitochondrial membrane fusion is a process essential for the maintenance of the structural integrity of the organelle. Since mitochondria are bounded by a double membrane, they face the challenge of fusing four membranes in a coordinated manner. We provide evidence that this is achieved by coupling of the mitochondrial outer and inner membranes by the mitochondrial fusion machinery. Fzo1, the first known mediator of mitochondrial fusion, spans the outer membrane twice, exposing a short loop to the intermembrane space. The presence of the intermembrane space segment is required for the localization of Fzo1 in sites of tight contact between the mitochondrial outer and inner membranes. Mutations in the intermembrane space domain of yeast Fzo1 relieve the association with the inner membrane. This results in a loss of function of the protein in vivo. We propose that the mitochondrial fusion machinery forms membrane contact sites that mediate mitochondrial fusion. A fusion machinery that is in contact with both mitochondrial membranes appears to be functionally important for coordinated fusion of four mitochondrial membranes.     

Mitochondrial morphology and dynamics: actors, mechanisms and functions

Mitochondria are dynamic organelles that continuously move, fuse and divide. Their overall morphology, ranging from a filamentous network to a collection of isolated dots, is determined by fusion-fission equilibrium, which depends on the cellular and physiological context. The machineries of fusion and fission, that are conserved throughout evolution, include three large GTPases of the dynamin-superfamily: Dnm1/DRP1 - involved in fission?- as well as Fzo1/MFN and Mgm1/OPA1?- required for fusion. While the activities, mecanisms and regulations of mitochondrial fusion and fission machineries continue to be unravelled, the relevance of mitochondrial dynamics is witnessed by their impact on organelle functions, cell survival and cell differenciation, their requirement for embryonic development and their involvement in neurological diseases.     

The intramitochondrial dynamin-related GTPase,Mgm1p,is a component of a protein complex that mediates mitochondrial fusion

A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Deltamgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion.     

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.     

Mitochondrial fusion in human cells is efficient,requires the inner membrane potential,and is mediated by mitofusins

Mitochondrial fusion remains a largely unknown process despite its observation by live microscopy and the identification of few implicated proteins. Using green and red fluorescent proteins targeted to the mitochondrial matrix, we show that mitochondrial fusion in human cells is efficient and achieves complete mixing of matrix contents within 12 h. This process is maintained in the absence of a functional respiratory chain, despite disruption of microtubules or after significant reduction of cellular ATP levels. In contrast, mitochondrial fusion is completely inhibited by protonophores that dissipate the inner membrane potential. This inhibition, which results in rapid fragmentation of mitochondrial filaments, is reversible: small and punctate mitochondria fuse to reform elongated and interconnected ones upon withdrawal of protonophores. Expression of wild-type or dominant-negative dynamin-related protein 1 showed that fragmentation is due to dynamin-related protein 1-mediated mitochondrial division. On the other hand, expression of mitofusin 1 (Mfn1), one of the human Fzo homologues, increased mitochondrial length and interconnectivity. This process, but not Mfn1 targeting, was dependent on the inner membrane potential, indicating that overexpressed Mfn1 stimulates fusion. These results show that human mitochondria represent a single cellular compartment whose exchanges and interconnectivity are dynamically regulated by the balance between continuous fusion and fission reactions.     

A Mutation Associated with CMT2A Neuropathy Causes Defects in Fzo1 GTP Hydrolysis,Ubiquitylation, and Protein Turnover

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.     

Regulation of mitochondrial fusion and division

In many organisms, ranging from yeast to humans, mitochondria fuse and divide to change their morphology in response to a multitude of signals. During the past decade, work using yeast and mammalian cells has identified much of the machinery required for fusion and division, including the dynamin-related GTPases--mitofusins (Fzo1p in yeast) and OPA1 (Mgm1p in yeast) for fusion and Drp1 (Dnm1p) for division. However, the mechanisms by which cells regulate these dynamic processes have remained largely unknown. Recent studies have uncovered regulatory mechanisms that control the activity, assembly, distribution and stability of the key components for mitochondrial fusion and division. In this review, we discuss how mitochondrial dynamics are controlled and how these events are coordinated with cell growth, mitosis, apoptosis and human diseases.     

Mitochondrial morphogenesis: Fusion factor for fly fertility

The Drosophila Fzo protein is required for mitochondrial fusion during a key step in sperm formation. This novel, membrane-bound GTPase defines a family of proteins that may mediate mitochondrial fusion in a variety of eukaryotic cells.     

Domain interactions within Fzo1 oligomers are essential for mitochondrial fusion

Mitofusins are conserved GTPases essential for the fusion of mitochondria. These mitochondrial outer membrane proteins contain a GTPase domain and two or three regions with hydrophobic heptad repeats, but little is known about how these domains interact to mediate mitochondrial fusion. To address this issue, we have analyzed the yeast mitofusin Fzo1p and find that mutation of any of the three heptad repeat regions (HRN, HR1, and HR2) leads to a null allele. Specific pairs of null alleles show robust complementation, indicating that functional domains need not exist on the same molecule. Biochemical analysis indicates that this complementation is due to Fzo1p oligomerization mediated by multiple domain interactions. Moreover, we find that two non-overlapping protein fragments, one consisting of HRN/GTPase and the other consisting of HR1/HR2, can form a complex that reconstitutes Fzo1p fusion activity. Each of the null alleles disrupts the interaction of these two fragments, suggesting that we have identified a key interaction involving the GTPase domain and heptad repeats essential for fusion.     

An RNAi screen for mitochondrial proteins required to maintain the morphology of the organelle in Caenorhabditis elegans

Mitochondria are dynamic organelles that frequently divide and fuse together, resulting in the formation of intracellular tubular networks. In yeast and mammals, several factors including Drp1/Dnm1 and Mfn/Fzo1 are known to regulate mitochondrial morphology by controlling membrane fission or fusion. Here, we report the systematic screening of Caenorhabditis elegans mitochondrial proteins required to maintain the morphology of the organelle using an RNA interference feeding library. In C. elegans body wall muscle cells, mitochondria usually formed tubular structures and were severely fragmented by the mutation in fzo-1 gene, indicating that the body wall muscle cells are suitable for monitoring changes in mitochondrial morphology due to gene silencing. Of 719 genes predicted to code for most of mitochondrial proteins, knockdown of >80% of them caused abnormal mitochondrial morphology, including fragmentation and elongation. These findings indicate that most fundamental mitochondrial functions, including metabolism and oxidative phosphorylation, are necessary for maintenance of the tubular networks as well as membrane fission and fusion. This is the first evidence that known mitochondrial activities are prerequisite for regulating the morphology of the organelle. Furthermore, 88 uncharacterized or poorly characterized genes were found in the screening to be implicated in mitochondrial morphology.