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Mitochondrial membrane fusion
Biochimica et Biophysica Acta 1641 (2003) 195 – 202 www.bba-direct.com
Review
Mitochondrial membrane fusion Benedikt Westermann * Institut fu¨r Physiologische Chemie der Universita¨t Mu¨nchen, Butenandtstr. 5, 81377 Mu¨nchen, Germany Received 31 October 2002; accepted 7 February 2003
Abstract Mitochondrial fusion has been observed in a great variety of organisms from yeast to man. It serves to mix and unify the mitochondrial compartment and plays roles in cellular aging, cell development, energy dissipation and mitochondrial DNA inheritance. Large GTPases in the mitochondrial outer membrane, termed Fzo or mitofusins, have been identified as key components of the mitochondrial fusion machinery in yeast, flies and mammalian cells. Recent studies in yeast suggest an involvement of a dynamin-related protein in the intermembrane space. Additional components have been identified by genetic screens. These findings suggest a unique and evolutionarily conserved mechanism for mitochondrial membrane fusion. D 2003 Elsevier B.V. All rights reserved. Keywords: Dynamin; Fzo; Membrane fusion; Mitochondrion; Mitofusin; Organelle biogenesis
1. Introduction The first microscopic observations of mitochondria date back more than 100 years. Since then, we have learned a lot about their metabolic functions. About 50 years ago, mitochondria were recognized as the cellular power plants, and in the following decades they were identified as the sites for the biosynthesis of many metabolites, iron/sulfur cluster assembly and other metabolic processes [1]. Research on mitochondria gained much interest in recent years because their functions in apoptosis [2,3], aging [4,5], calcium homeostasis [6,7] and cell development [8] were recognized. Moreover, mitochondria have been suggested to play important roles in the pathogenesis of a number of diseases including Alzheimer’s [9] and Parkinson’s disease [10] and cancer [11,12]. However, the cellular mechanisms that govern mitochondrial behavior are still only poorly understood. In many cell types the mitochondrial compartment forms a giant interconnected reticulum. In budding yeast, for example, 1 to 10 organelles per cell form a branched tubular network located below the cell cortex [13 – 15], and interconnected mitochondrial networks have been described * Tel.: +49-89-2180-77122; fax: +49-89-2180-77093. E-mail address: [email protected] (B. Westermann). URL: http://www.med.uni-muenchen.de/physiolchem/westermann. html. 0167-4889/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-4889(03)00091-0
in fibroblasts in culture [16], rat liver cells [17] and a variety of unicellular organisms [18]. Cytological and genetic studies showed that establishment and maintenance of the mitochondrial reticulum depend on a balanced frequency of organellar fusion and fission events in a great variety of different cell types from yeast to mammalian cells [19 –23]. There is accumulating evidence that the dynamic behavior of mitochondria is crucial for many cellular functions (summarized in Fig. 1). Fusion of mitochondria is required for the inheritance of mitochondrial DNA in yeast [20,24,25], it is a prerequisite for the complementation of oxidatively damaged mitochondrial gene products as a defence mechanism against cellular aging [26,27], and it plays an essential role in cell developmental processes, as for example spermatogenesis in flies [8]. Furthermore, the establishment of intracellular mitochondrial networks by mitochondrial fusion is important for the transmission of energy and calcium signals [28]. On the other hand, morphologically separated and functionally distinct mitochondria are required during apoptosis [29,30]. Several reviews discussing the dynamic behavior of mitochondria have been published in recent years [31,32], some focusing on the molecular components of mitochondrial distribution and morphology in yeast [33 –36] or in filamentous fungi [37], and others concentrating on the machinery of mitochondrial division [38 –40] or the cellular role of mitochondrial fusion [41,42]. Here, I will review the genetic and biochemical analyses that have identified and
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suggests that members of the Fzo protein family, also termed mitofusins, are central components of this fusion machinery, as discussed below.
3. The Fzo protein family/mitofusins 3.1. Discovery of the fzo gene
Fig. 1. Size, shape and interconnectivity of mitochondria are determined by fusion and fission. By shifting the equilibrium to either side, mitochondria can adapt to various physiological challenges. See text for details.
functionally characterized the molecular components involved in mitochondrial membrane fusion.
2. Mitochondria possess a unique membrane fusion machinery The molecular components mediating mitochondrial fusion are not related to other intracellular membrane fusion factors. In particular, the key components of the fusion machinery of the secretory pathway, such as NSF and SNARE proteins [43], are apparently not required for mitochondrial fusion. Temperature-sensitive mutations of yeast NSF and its homologs have no effect on mitochondrial fusion [20], and the SNARE proteins from yeast, which are all known from its genome sequence, are exclusively localized to the organelles of the secretory and endocytic pathways [44]. There is only one report suggesting a localization of a SNARE protein on mitochondria. A splice isoform of vesicle-associated membrane protein-1, VAMP-1B, was found associated with mitochondria upon overexpression from a heterologous promoter in human endothelial cells [45]. However, it is unclear whether a localization of SNAREs to mitochondria is common in mammalian cells, and whether they are involved in mitochondrial fusion. Instead, mitochondria-localized SNAREs could play a role in establishing contact to heterotypic membranes, which might be important for example for lipid transfer between cellular compartments, or for other inter-organellar communication processes. It is conceivable that the membrane fusion machinery of the endosymbiotic ancestors of mitochondria evolved independently of the complex system of SNARE-like fusion proteins that governed vesicular trafficking pathways of ancient eukaryotic cells. As double-membrane-bounded organelles, mitochondria require a fusion machinery that faithfully fuses four membranes in a coordinated manner, a challenge that obviously cannot be met by SNARE proteins. Thus, nature had to develop a novel machinery mediating homotypic fusion of mitochondria. Accumulating evidence
Mitochondria undergo dramatic morphogenetic rearrangements during sperm development. In early spermatids of Drosophila melanogaster, mitochondria aggregate beside the nuclei, and then fuse into two giant organelles that wrap around each other to build a large spherical structure, termed Nebenkern. This stage of spermatid development is termed ‘onion stage’ because the Nebenkern resembles an onion slice when viewed in cross section by electron microscopy. During further development, the two mitochondrial derivatives unfurl from each other and elongate beside the flagellar base, where they provide energy for the vigorous movement of the mature sperm cell [46]. The first known component of the mitochondrial membrane fusion machinery, the Fzo protein, has been identified by molecular genetic analysis of a mutant defective in Nebenkern formation, the fuzzy onions (fzo) mutant. Early spermatids of fzo mutant flies display misshapen Nebenkerns consisting of many small mitochondria that are wrapped around each other and form a structure resembling ‘fuzzy onions’. Block of mitochondrial fusion during Nebenkern formation in fzo mutants leads to male sterility. Mapping of the mutant allele and complementation studies identified the fzo gene [8]. 3.2. Evolutionary conservation of the Fzo protein family Fzo is the founding member of a novel protein family of large GTPases located in the mitochondrial outer membrane [8]. Fzo protein family members, also termed ‘mitofusins’, have been conserved during evolution throughout the fungal and animal kingdoms. Homologs of Fzo have been functionally characterized in the budding yeast Saccharomyces cerevisiae and in human cells (see below). As yet uncharacterized genes encoding related proteins can be found in the genomes of the nematode worm Caenorhabditis elegans, the puffer fish Fugu rubripes, the filamentous fungus Neurospora crassa and the fission yeast Schizosaccharomyces pombe (Table 1 and Fig. 2). 3.3. Expression pattern of fzo-related genes The Fzo1 protein of budding yeast is uniformly distributed on the mitochondrial compartment. Fzo1p levels do not change during mitotic growth, mating or meiosis [25]. This is consistent with the observation that mitochondria of vegetatively growing wild-type yeast fuse and divide continuously during all stages of the cell cycle at an average rate of approximately one fusion event per cell in 2 min [20].
B. Westermann / Biochimica et Biophysica Acta 1641 (2003) 195–202 Table 1 Known and predicted Fzo family members Protein Expressiona
Organism
Gene
Caenorhabditis elegans Drosophila melanogaster
ZK1248.14
Drosophila melanogaster
dmfn
Fugu rubripes Fugu rubripes Homo sapiens
JGI 23164 JGI 5215 mfn1
Mfn1
Homo sapiens
mfn2
Mfn2
fuzzy onions (fzo)
n.d. Fzo
Neurospora NCU00436.1 crassa Saccharomyces FZO1 Fzo1p cerevisiae Schizosaccharomyces SPBC1706.03 pombe a
References
spermatocytes, early spermatids broad expression pattern n.d. n.d. ubiquitous expression ubiquitous expression n.d.
[8,49]
constitutive expression n.d.
[24,25]
[49]
[47,52] [47,52]
n.d., not determined.
Interestingly, higher metazoa such as insects, fish and mammals have two fzo-related genes. Both human isoforms, Mfn1 and Mfn2, are expressed ubiquitously and at similar levels in a variety of tissues including brain, fibroblasts, heart, kidney, liver, muscle and testis [47]. It is unknown whether the two mammalian mitofusins display different activities, or whether they act in a synergistic manner. Under normal conditions, human mitofusins appear evenly distributed on the mitochondrial surface [47] where they mediate efficient mitochondrial fusion [23]. In apoptotic cells, however, Mfn2 is found in punctate structures associated with the mitochondrial fission protein Drp1 and the pro-apoptotic
Fig. 2. Homology tree of the Fzo protein family. The tree was constructed using DNAMAN software (Lynnon BioSoft, Vaudreuil, Canada). Proteins are indicated by their name or by their genome annotation number. See Table 1 for references.
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factor Bax [48]. It will be interesting to see in the future whether this behavior reflects a yet unknown role of mitofusins in apoptosis. The two mitofusins of Drosophila, Dmfn and Fzo, are differentially expressed. Transcripts of the dmfn gene are detected in both adult males and females, and at various stages during embryogenesis [49]. Furthermore, expressed sequence tags are found in libraries originated from embryo, head and ovary [47]. In contrast, expression of the fuzzy onions gene is restricted to early stage spermatids late in meiosis. The protein can be detected just at the time when mitochondrial fusion occurs to build the Nebenkern, and later disappears [8,49]. Thus, the protein encoded by the fuzzy onions gene apparently exerts a highly specialized function during sperm development, while the product of the dmfn gene might play a more general role in controlling mitochondrial morphology in the fly. 3.4. Domain structure and topology of Fzo family members The molecular weight of Fzo-related proteins ranges from 81 kDa (Drosophila Fzo) to 101 kDa (predicted protein of Neurospora). All Fzo family members have a similar domain structure and topology (Fig. 3). The N-terminal part contains a conserved predicted GTPase domain. Near the C terminus, there are two transmembrane segments which are separated
Fig. 3. Domain structure and topology of Fzo proteins. Intramolecular coiled-coil pairing is inferred from data presented in Ref. [47]. It should be noted that intermolecular coiled-coil formation, or the formation of multiple helix bundles assembled from coiled-coil domains of different molecules, is equally well possible. OM, mitochondrial outer membrane; IM, mitochondrial inner membrane; TM, transmembrane domain.
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by a short stretch rich in positively charged amino acid residues. The transmembrane segments are flanked by predicted coiled-coil domains. The fungal homologs and the predicted C. elegans protein contain, in addition, a third predicted coiled-coil region located N-terminal of the GTPase domain [8,25,47]. Fzo proteins are integral proteins of the mitochondrial outer membrane. Both the large N-terminal part and the smaller C-terminal tail are exposed to the cytosol [24,25, 47,50]. The intermembrane space loop is required for a localization of yeast Fzo1p in sites of tight contact between the mitochondrial outer and inner membranes and presumably interacts with as yet unknown inner membrane proteins [50]. Yeast Fzo1p has been shown to assemble into a high molecular weight complex of 800 kDa of yet unknown composition [24]. 3.5. Knock out mutants of fzo-related genes The analysis of deletion mutants in yeast proved that Fzo1p is a key component of the mitochondrial fusion machinery. Yeast mutants lacking the FZO1 gene harbor fragmented mitochondria because fusion is blocked while fission is going on. As a secondary consequence of aberrant mitochondrial morphology, Dfzo1 mutants lose their mitochondrial genome and therefore acquire a respiration-deficient growth phenotype [24,25]. Intriguingly, fragmentation of mitochondria and loss of mitochondrial DNA in Dfzo1 mutants can be suppressed by deletion of the DNM1 gene, which encodes a key component of the mitochondrial division machinery [21,22]. This suggests that mitochondrial shape is controlled by a balance between fusion, requiring Fzo1p, and division, mediated by Dnm1p. Direct evidence for a role of Fzo1p in mitochondrial fusion was obtained by using a microscopic assay that allows visualization of mitochondrial fusion and content mixing in yeast zygotes. Upon mating of haploid wild-type cells preloaded with different fluorescent markers in the mitochondrial matrix, the fluorescent labels immediately intermix in zygotes, indicating fusion of the parental mitochondrial membranes [20]. A conditional mutant, fzo1-1, exhibits rapid and extensive fragmentation of mitochondria already 10 min after shift to the nonpermissive temperature. When fzo1-1 cells are crossed at the nonpermissive temperature, mitochondrial networks fragment and fail to fuse. Fusion is also strongly reduced in matings between fzo1-1 and wild-type cells, suggesting that functional Fzo1p is required on mitochondria of both haploid parents [25]. Mice lacking either one of the mfn1 or mfn2 genes die early during embryonic development. Mfn2 mutant mice show a specific defect in placental development, which might be the primary reason for embryonic lethality. Interestingly, a placental defect was not observed in Mfn1 mutant mice despite expression of Mfn1 in the placenta. Both Mfn1- and Mfn2-deficient cells harbor fragmented mitochondria that are deficient in mitochondrial fusion in tissue culture cells [51].
3.6. Overexpression of Fzo proteins A role of mammalian mitofusins in determining mitochondrial shape was first proposed based on overexpression studies in tissue culture cells. Transfected HeLa or COS-7 cells highly overexpressing Mfn2 exhibit massive perinuclear clustering of mitochondria [47,52]. Ultrastructural examination of these cells by electron microscopy revealed spherical or ovoid mitochondria with increased diameter and swollen cristae. Intriguingly, the membranes of apposing mitochondria were in close contact without merging [47]. This suggests that fusion might be blocked at the stage of adherence or docking of the organelles, possibly by accumulation of unproductive fusion intermediates. 3.7. Structure – function studies Functional analysis of a number of site-specific mutants in different organisms shed light on structure – function relationships of Fzo-related proteins. The GTPase domain contains four conserved motifs, G1 –G4, which are also found in other GTPases, where they are required for GTP binding and hydrolysis [53]. Most mutations of conserved amino acid residues within these motifs result in a complete loss of function of Fzo proteins. GTPase mutants of Drosophila Fzo are male sterile and defective in Nebenkern formation [8]; GTPase mutants of yeast Fzo1p harbor fragmented mitochondria and lose their mitochondrial genome [25]; and GTPase mutants of human Mfn2 are unable to generate extended mitochondrial networks under conditions of compromised mitochondrial fission [52]. These observations suggest that GTP binding and/or hydrolysis by Fzo is essential for mitochondrial fusion. On the other hand, a functional GTPase is not required for mitochondrial clustering by overexpression of Mfn2 in mammalian cells [47,52]. In contrast, a role in mitochondrial clustering was suggested for coiled-coil regions because a truncation mutant lacking the C-terminal coiled-coil of human Mfn2 showed a strong reduction of clustering [52]. Furthermore, coiled-coil regions are apparently important for inter- or intramolecular interactions of Fzo proteins. A C-terminal Mfn2 fragment covering the transmembrane segments and the C-terminal domain is able to recruit an otherwise soluble N-terminal fragment lacking the transmembrane segments to mitochondria, and is able to induce clustering. Mitochondrial localization and clustering depend on the coiled-coil domains in both fragments [47]. Several studies have demonstrated that the transmembrane segments and surrounding regions are important for targeting of Fzo proteins to the mitochondrial outer membrane [47,50,52]. Moreover, the intermembrane space loop between the transmembrane regions of Fzo1p was shown to be important for the protein’s function in yeast. Insertion of a flexible linker between both transmembrane domains abolishes the contact of the mutant protein to the inner membrane and causes fragmentation of mitochondria [50].
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3.8. Models for Fzo function The observations described above can be integrated into a—yet rather hypothetical—model of Fzo function. It is conceivable that an early step of mitochondrial fusion requires docking of two organelles approaching each other. This step might involve intermolecular interactions between Fzo proteins on opposite mitochondria mediated by coiledcoil domains. In addition, inter- or intramolecular coiled-coil interactions of molecules of the same membrane might be important. It has been speculated that Fzo coiled-coils form a four helix bundle [47], as it is also seen in SNARE proteins [54]. If such a multiple helix bundle is assembled from molecules of opposite organelles, it could act directly as a fusogen of the outer membrane. A putative self-interaction (as pictured in Fig. 3) is only one out of several possible assembly states of Fzo. Moreover, it cannot be excluded that other domains of Fzo and/or other proteins are involved. The GTPase could play a regulatory role in modulating the activity of Fzo or other components of the fusion machinery. Alternatively, the GTPase could provide biomechanical energy that could be used directly to overcome the energy barrier of membrane fusion. In the latter case, Fzo GTPases would act as mechanoenzymes similar to dynamin-like proteins [55,56]. Finally, the intermembrane space loop connects the outer membrane fusion complex to components in the inner membrane or intermembrane space to coordinate fusion of the mitochondrial outer and inner membranes [50]. Although this mode of action is compatible with all available data, many aspects of Fzo function are yet rather speculative, and many important questions remain unanswered. For example, it has not been demonstrated that Fzolike proteins act directly as fusogens for the outer membrane. It is equally well possible that Fzo family members are key regulators acting on yet unknown proteins that mediate mixing of the lipid bilayers. It can be expected that uncovering the composition of the Fzo1p-containing 800kDa complex in the outer membrane [24] and identification of putative interaction partners in the inner membrane [50] will help to clarify these issues in the future.
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was first identified in a screen for genes required for mitochondrial genome maintenance, MGM [58]. Later it was demonstrated that mgm1 mutants harbor abnormal mitochondria [59,60]. Examination of temperature-sensitive mgm1 mutants identified fragmentation of mitochondrial reticuli as the primary phenotype associated with loss of Mgm1p function [57]. Intriguingly, mitochondrial fragmentation and loss of mitochondrial DNA in mgm1 mutants are suppressed by mutations of components of mitochondrial fission [57,61 – 63], and mitochondrial fusion is blocked in zygotes lacking Mgm1p [36,64]. These phenotypes are very similar to that observed in fzo1 mutant cells, suggesting that Mgm1p might also be required for mitochondrial fusion. Moreover, Mgm1p was found to physically interact with fusion factors in yeast, Fzo1p and Ugo1p [64]. However, these interactions might be only transient, and their mechanistic significance is not fully understood. The intramitochondrial localization of Mgm1p and its orthologs has been a matter of debate [57,60,65]. Recent results are in favor of a localization in the intermembrane space [36,64,66]. The precise role of Mgm1-like proteins in mitochondrial morphogenesis is not known. Suggested functions include reorganization of mitochondria following a change in carbon source [59], anchoring mitochondrial DNA to the inner membrane [67], local influence on the structural properties of mitochondrial membranes [60], fission and/or remodelling of the inner membrane [57,65], formation or stabilization of inner membrane cristae [57,65,66], and coordination of fusion of the mitochondrial membranes [64]. Thus, it is at present unclear whether the role of Mgm1-related proteins in mitochondrial fusion is direct or indirect. Elucidation of the molecular role of Mgm1p and its orthologues will be of considerable interest because OPA1, the human homolog of MGM1, has recently been identified as the gene responsible for dominant optical atrophy (DOA), the most common form of inherited optic neuropathy [68,69]. This disease results in a progressive loss of retinal ganglion cells that is associated with childhood and adult blindness and may represent the first case of a mitochondrial disease caused by an alteration of mitochondrial dynamics [70].
4. A role of dynamin-like proteins in mitochondrial fusion? 5. Additional lessons from yeast Dynamin is a mechanoenzyme that mediates the release of clathrin-coated vesicles from the plasma membrane during endocytosis. Recently, a number of proteins related to this large GTPase have been implicated in a variety of membrane trafficking events, including maintenance of mitochondrial morphology [38,55,56]. In particular, dynamin-like proteins assembling on the mitochondrial surface are thought to mediate mitochondrial division by severing the mitochondrial membranes [36,38 – 40]. A role in mitochondrial fusion has been proposed for the dynamin-like protein Mgm1p in yeast [57]. This component
Genetic screens in yeast have identified additional components that might play important roles in mitochondrial fusion. Ugo1p is a novel protein of the mitochondrial outer membrane. The phenotype of yeast cells lacking Ugo1p resembles that of fzo1 and mgm1 mutants. Dugo1 mutants harbor fragmented mitochondria, lose mitochondrial DNA and fail to fuse mitochondria in zygotes [62]. As Ugo1p does not localize with Fzo1p to sites of close contact between the mitochondrial outer and inner membranes, and as evidence for an interaction of Ugo1p and Fzo1p
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could not be obtained, it is likely that these two proteins do not permanently interact. Thus, they presumably mediate distinct steps in mitochondrial fusion [62]. A comprehensive genome-wide screen for yeast mutants with defective mitochondrial distribution and morphology recently uncovered a number of novel genes involved in mitochondrial morphogenesis. A deletion mutant library covering 4794 nonessential yeast genes was systematically screened with mitochondria-specific fluorescent probes. Ten previously uncharacterized genes were identified to be essential for establishment and maintenance of a wild type-like mitochondrial morphology [71]. Some of these novel mdm mutants displayed fragmented mitochondria, suggesting a defect in mitochondrial fusion. This class of mutants includes mdm30 and mdm37. Certain motifs within the Mdm30p and Mdm37p sequences point to a possible role of these proteins as regulators of mitochondrial fusion. The Mdm30p sequence contains an F-box motif which is commonly found in subunits of Skp1-Cdc53-F-box protein (SCF) ubiquitin ligases [72]. Cells lacking Mdm30p contain highly aggregated or fragmented mitochondria and fail to fuse mitochondria in zygotes. The protein level of Fzo1p is regulated by Mdm30p. Thus, Mdm30p controls mitochondrial shape by regulating the steady state level of Fzo1 and apparently links the ubiquitin/26S proteasome system to mitochondria [73]. Mdm37p shares homology with the rhomboid protein of Drosophila, which is involved in proteolytic activation of factors of the epidermal growth factor signalling pathway [74]. Recently, yeast Mdm37p (alternative name Pcp1p) was shown to be involved in proteolytic processing of the enzyme cytochrome c peroxidase in the intermembrane space [75]. It is tempting to speculate that Mdm37p is involved in proteolytic activation of mitochondrial fusion factors in a similar manner.
6. Perspectives Fusion of mitochondria requires faithful merging of four membranes that must be intimately coordinated with organellar fission processes. Given the complexity of this reaction, it can be expected that several important proteins contributing to this process remain to be discovered. These will likely include components of the fusion machinery in the mitochondrial inner membrane, regulatory proteins and maybe additional factors in the outer membrane and intermembrane space. Dissecting the biochemical roles of known and novel players of mitochondrial fusion will require the development of in vitro assays and reconstitution of individual steps of the fusion process. Detailed analysis of knock-out mutants in animal model systems will teach us more about the role of mitochondrial fusion in cell development, apoptosis, aging and physiology. Clearly, the elucidation of the cellular role and molecular mechanisms of
mitochondrial fusion will remain a fascinating area of cell biological research for the coming years. Acknowledgements I thank the members of my laboratory for their invaluable work, Walter Neupert for continuous support, Hannes Herrmann for many stimulating discussions and for critical comments on the manuscript, and the Deutsche Forschungsgemeinschaft for support through grant WE 2174/2-3. References [1] I.E. Scheffler, A century of mitochondrial research: achievements and perspectives, Mitochondrion 1 (2001) 3 – 31. [2] S.A. Susin, N. Zamzami, G. Kroemer, Mitochondria as regulators of apoptosis: doubt no more, Biochim. Biophys. Acta 1366 (1998) 151 – 165. [3] X. Wang, The expanding role of mitochondria in apoptosis, Genes Dev. 15 (2001) 2922 – 2933. [4] G. Lenaz, Role of mitochondria in oxidative stress and ageing, Biochim. Biophys. Acta 1366 (1998) 53 – 67. [5] S. Raha, B.H. Robinson, Mitochondria, oxygen free radicals, disease and ageing, Trends Biochem. Sci. 25 (2000) 502 – 508. [6] F. Ichas, J.P. Mazat, From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low-to high-conductance state, Biochim. Biophys. Acta 1366 (1998) 33 – 50. [7] G.A. Rutter, R. Rizzuto, Regulation of mitochondrial metabolism by ER Ca2 + release: an intimate connection, Trends Biochem. Sci. 25 (2000) 215 – 221. [8] K.G. Hales, M.T. Fuller, Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase, Cell 90 (1997) 121 – 129. [9] E. Bonilla, K. Tanji, M. Hirano, T.H. Vu, S. DiMauro, E.A. Schon, Mitochondrial involvement in Alzheimer’s disease, Biochim. Biophys. Acta 1410 (1999) 171 – 182. [10] A.H. Schapira, Mitochondrial involvement in Parkinson’s disease, Huntington’s disease, hereditary spastic paraplegia and Friedreich’s ataxia, Biochim. Biophys. Acta 1410 (1999) 159 – 170. [11] L.R. Cavalli, B.C. Liang, Mutagenesis, tumorigenicity, and apoptosis: are the mitochondria involved? Mutat. Res. 398 (1998) 19 – 26. [12] V.R. Fantin, M.J. Berardi, L. Scorrano, S.J. Korsmeyer, P. Leder, A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth, Cancer Cell 2 (2002) 29 – 42. [13] H.-P. Hoffmann, C.J. Avers, Mitochondrion of yeast: ultrastructural evidence for one giant, branched organelle per cell, Science 181 (1973) 749 – 750. [14] B. Stevens, Mitochondrial structure, in: E.W. Strathern, E.W. Jones, J.R. Broach (Eds.), The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1981, pp. 471 – 504. [15] A. Egner, S. Jakobs, S.W. Hell, Fast 100 nm resolution 3D-microscope reveals structural plasticity of mitochondria in live yeast, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 3370 – 3375. [16] L.V. Johnson, M.L. Walsh, L.B. Chen, Localization of mitochondria in living cells with rhodamine 123, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 990 – 994. [17] J.T. Brandt, A.P. Martin, F.V. Lucas, M.L. Vorbeck, The structure of rat liver mitochondria: a reevaluation, Biochem. Biophys. Res. Commun. 59 (1974) 1097 – 1104. [18] J. Bereiter-Hahn, Behavior of mitochondria in the living cell, Int. Rev. Cyt. 122 (1990) 1 – 63.
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Review
Mitochondrial membrane fusion Benedikt Westermann * Institut fu¨r Physiologische Chemie der Universita¨t Mu¨nchen, Butenandtstr. 5, 81377 Mu¨nchen, Germany Received 31 October 2002; accepted 7 February 2003
Abstract Mitochondrial fusion has been observed in a great variety of organisms from yeast to man. It serves to mix and unify the mitochondrial compartment and plays roles in cellular aging, cell development, energy dissipation and mitochondrial DNA inheritance. Large GTPases in the mitochondrial outer membrane, termed Fzo or mitofusins, have been identified as key components of the mitochondrial fusion machinery in yeast, flies and mammalian cells. Recent studies in yeast suggest an involvement of a dynamin-related protein in the intermembrane space. Additional components have been identified by genetic screens. These findings suggest a unique and evolutionarily conserved mechanism for mitochondrial membrane fusion. D 2003 Elsevier B.V. All rights reserved. Keywords: Dynamin; Fzo; Membrane fusion; Mitochondrion; Mitofusin; Organelle biogenesis
1. Introduction The first microscopic observations of mitochondria date back more than 100 years. Since then, we have learned a lot about their metabolic functions. About 50 years ago, mitochondria were recognized as the cellular power plants, and in the following decades they were identified as the sites for the biosynthesis of many metabolites, iron/sulfur cluster assembly and other metabolic processes [1]. Research on mitochondria gained much interest in recent years because their functions in apoptosis [2,3], aging [4,5], calcium homeostasis [6,7] and cell development [8] were recognized. Moreover, mitochondria have been suggested to play important roles in the pathogenesis of a number of diseases including Alzheimer’s [9] and Parkinson’s disease [10] and cancer [11,12]. However, the cellular mechanisms that govern mitochondrial behavior are still only poorly understood. In many cell types the mitochondrial compartment forms a giant interconnected reticulum. In budding yeast, for example, 1 to 10 organelles per cell form a branched tubular network located below the cell cortex [13 – 15], and interconnected mitochondrial networks have been described * Tel.: +49-89-2180-77122; fax: +49-89-2180-77093. E-mail address: [email protected] (B. Westermann). URL: http://www.med.uni-muenchen.de/physiolchem/westermann. html. 0167-4889/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-4889(03)00091-0
in fibroblasts in culture [16], rat liver cells [17] and a variety of unicellular organisms [18]. Cytological and genetic studies showed that establishment and maintenance of the mitochondrial reticulum depend on a balanced frequency of organellar fusion and fission events in a great variety of different cell types from yeast to mammalian cells [19 –23]. There is accumulating evidence that the dynamic behavior of mitochondria is crucial for many cellular functions (summarized in Fig. 1). Fusion of mitochondria is required for the inheritance of mitochondrial DNA in yeast [20,24,25], it is a prerequisite for the complementation of oxidatively damaged mitochondrial gene products as a defence mechanism against cellular aging [26,27], and it plays an essential role in cell developmental processes, as for example spermatogenesis in flies [8]. Furthermore, the establishment of intracellular mitochondrial networks by mitochondrial fusion is important for the transmission of energy and calcium signals [28]. On the other hand, morphologically separated and functionally distinct mitochondria are required during apoptosis [29,30]. Several reviews discussing the dynamic behavior of mitochondria have been published in recent years [31,32], some focusing on the molecular components of mitochondrial distribution and morphology in yeast [33 –36] or in filamentous fungi [37], and others concentrating on the machinery of mitochondrial division [38 –40] or the cellular role of mitochondrial fusion [41,42]. Here, I will review the genetic and biochemical analyses that have identified and
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suggests that members of the Fzo protein family, also termed mitofusins, are central components of this fusion machinery, as discussed below.
3. The Fzo protein family/mitofusins 3.1. Discovery of the fzo gene
Fig. 1. Size, shape and interconnectivity of mitochondria are determined by fusion and fission. By shifting the equilibrium to either side, mitochondria can adapt to various physiological challenges. See text for details.
functionally characterized the molecular components involved in mitochondrial membrane fusion.
2. Mitochondria possess a unique membrane fusion machinery The molecular components mediating mitochondrial fusion are not related to other intracellular membrane fusion factors. In particular, the key components of the fusion machinery of the secretory pathway, such as NSF and SNARE proteins [43], are apparently not required for mitochondrial fusion. Temperature-sensitive mutations of yeast NSF and its homologs have no effect on mitochondrial fusion [20], and the SNARE proteins from yeast, which are all known from its genome sequence, are exclusively localized to the organelles of the secretory and endocytic pathways [44]. There is only one report suggesting a localization of a SNARE protein on mitochondria. A splice isoform of vesicle-associated membrane protein-1, VAMP-1B, was found associated with mitochondria upon overexpression from a heterologous promoter in human endothelial cells [45]. However, it is unclear whether a localization of SNAREs to mitochondria is common in mammalian cells, and whether they are involved in mitochondrial fusion. Instead, mitochondria-localized SNAREs could play a role in establishing contact to heterotypic membranes, which might be important for example for lipid transfer between cellular compartments, or for other inter-organellar communication processes. It is conceivable that the membrane fusion machinery of the endosymbiotic ancestors of mitochondria evolved independently of the complex system of SNARE-like fusion proteins that governed vesicular trafficking pathways of ancient eukaryotic cells. As double-membrane-bounded organelles, mitochondria require a fusion machinery that faithfully fuses four membranes in a coordinated manner, a challenge that obviously cannot be met by SNARE proteins. Thus, nature had to develop a novel machinery mediating homotypic fusion of mitochondria. Accumulating evidence
Mitochondria undergo dramatic morphogenetic rearrangements during sperm development. In early spermatids of Drosophila melanogaster, mitochondria aggregate beside the nuclei, and then fuse into two giant organelles that wrap around each other to build a large spherical structure, termed Nebenkern. This stage of spermatid development is termed ‘onion stage’ because the Nebenkern resembles an onion slice when viewed in cross section by electron microscopy. During further development, the two mitochondrial derivatives unfurl from each other and elongate beside the flagellar base, where they provide energy for the vigorous movement of the mature sperm cell [46]. The first known component of the mitochondrial membrane fusion machinery, the Fzo protein, has been identified by molecular genetic analysis of a mutant defective in Nebenkern formation, the fuzzy onions (fzo) mutant. Early spermatids of fzo mutant flies display misshapen Nebenkerns consisting of many small mitochondria that are wrapped around each other and form a structure resembling ‘fuzzy onions’. Block of mitochondrial fusion during Nebenkern formation in fzo mutants leads to male sterility. Mapping of the mutant allele and complementation studies identified the fzo gene [8]. 3.2. Evolutionary conservation of the Fzo protein family Fzo is the founding member of a novel protein family of large GTPases located in the mitochondrial outer membrane [8]. Fzo protein family members, also termed ‘mitofusins’, have been conserved during evolution throughout the fungal and animal kingdoms. Homologs of Fzo have been functionally characterized in the budding yeast Saccharomyces cerevisiae and in human cells (see below). As yet uncharacterized genes encoding related proteins can be found in the genomes of the nematode worm Caenorhabditis elegans, the puffer fish Fugu rubripes, the filamentous fungus Neurospora crassa and the fission yeast Schizosaccharomyces pombe (Table 1 and Fig. 2). 3.3. Expression pattern of fzo-related genes The Fzo1 protein of budding yeast is uniformly distributed on the mitochondrial compartment. Fzo1p levels do not change during mitotic growth, mating or meiosis [25]. This is consistent with the observation that mitochondria of vegetatively growing wild-type yeast fuse and divide continuously during all stages of the cell cycle at an average rate of approximately one fusion event per cell in 2 min [20].
B. Westermann / Biochimica et Biophysica Acta 1641 (2003) 195–202 Table 1 Known and predicted Fzo family members Protein Expressiona
Organism
Gene
Caenorhabditis elegans Drosophila melanogaster
ZK1248.14
Drosophila melanogaster
dmfn
Fugu rubripes Fugu rubripes Homo sapiens
JGI 23164 JGI 5215 mfn1
Mfn1
Homo sapiens
mfn2
Mfn2
fuzzy onions (fzo)
n.d. Fzo
Neurospora NCU00436.1 crassa Saccharomyces FZO1 Fzo1p cerevisiae Schizosaccharomyces SPBC1706.03 pombe a
References
spermatocytes, early spermatids broad expression pattern n.d. n.d. ubiquitous expression ubiquitous expression n.d.
[8,49]
constitutive expression n.d.
[24,25]
[49]
[47,52] [47,52]
n.d., not determined.
Interestingly, higher metazoa such as insects, fish and mammals have two fzo-related genes. Both human isoforms, Mfn1 and Mfn2, are expressed ubiquitously and at similar levels in a variety of tissues including brain, fibroblasts, heart, kidney, liver, muscle and testis [47]. It is unknown whether the two mammalian mitofusins display different activities, or whether they act in a synergistic manner. Under normal conditions, human mitofusins appear evenly distributed on the mitochondrial surface [47] where they mediate efficient mitochondrial fusion [23]. In apoptotic cells, however, Mfn2 is found in punctate structures associated with the mitochondrial fission protein Drp1 and the pro-apoptotic
Fig. 2. Homology tree of the Fzo protein family. The tree was constructed using DNAMAN software (Lynnon BioSoft, Vaudreuil, Canada). Proteins are indicated by their name or by their genome annotation number. See Table 1 for references.
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factor Bax [48]. It will be interesting to see in the future whether this behavior reflects a yet unknown role of mitofusins in apoptosis. The two mitofusins of Drosophila, Dmfn and Fzo, are differentially expressed. Transcripts of the dmfn gene are detected in both adult males and females, and at various stages during embryogenesis [49]. Furthermore, expressed sequence tags are found in libraries originated from embryo, head and ovary [47]. In contrast, expression of the fuzzy onions gene is restricted to early stage spermatids late in meiosis. The protein can be detected just at the time when mitochondrial fusion occurs to build the Nebenkern, and later disappears [8,49]. Thus, the protein encoded by the fuzzy onions gene apparently exerts a highly specialized function during sperm development, while the product of the dmfn gene might play a more general role in controlling mitochondrial morphology in the fly. 3.4. Domain structure and topology of Fzo family members The molecular weight of Fzo-related proteins ranges from 81 kDa (Drosophila Fzo) to 101 kDa (predicted protein of Neurospora). All Fzo family members have a similar domain structure and topology (Fig. 3). The N-terminal part contains a conserved predicted GTPase domain. Near the C terminus, there are two transmembrane segments which are separated
Fig. 3. Domain structure and topology of Fzo proteins. Intramolecular coiled-coil pairing is inferred from data presented in Ref. [47]. It should be noted that intermolecular coiled-coil formation, or the formation of multiple helix bundles assembled from coiled-coil domains of different molecules, is equally well possible. OM, mitochondrial outer membrane; IM, mitochondrial inner membrane; TM, transmembrane domain.
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by a short stretch rich in positively charged amino acid residues. The transmembrane segments are flanked by predicted coiled-coil domains. The fungal homologs and the predicted C. elegans protein contain, in addition, a third predicted coiled-coil region located N-terminal of the GTPase domain [8,25,47]. Fzo proteins are integral proteins of the mitochondrial outer membrane. Both the large N-terminal part and the smaller C-terminal tail are exposed to the cytosol [24,25, 47,50]. The intermembrane space loop is required for a localization of yeast Fzo1p in sites of tight contact between the mitochondrial outer and inner membranes and presumably interacts with as yet unknown inner membrane proteins [50]. Yeast Fzo1p has been shown to assemble into a high molecular weight complex of 800 kDa of yet unknown composition [24]. 3.5. Knock out mutants of fzo-related genes The analysis of deletion mutants in yeast proved that Fzo1p is a key component of the mitochondrial fusion machinery. Yeast mutants lacking the FZO1 gene harbor fragmented mitochondria because fusion is blocked while fission is going on. As a secondary consequence of aberrant mitochondrial morphology, Dfzo1 mutants lose their mitochondrial genome and therefore acquire a respiration-deficient growth phenotype [24,25]. Intriguingly, fragmentation of mitochondria and loss of mitochondrial DNA in Dfzo1 mutants can be suppressed by deletion of the DNM1 gene, which encodes a key component of the mitochondrial division machinery [21,22]. This suggests that mitochondrial shape is controlled by a balance between fusion, requiring Fzo1p, and division, mediated by Dnm1p. Direct evidence for a role of Fzo1p in mitochondrial fusion was obtained by using a microscopic assay that allows visualization of mitochondrial fusion and content mixing in yeast zygotes. Upon mating of haploid wild-type cells preloaded with different fluorescent markers in the mitochondrial matrix, the fluorescent labels immediately intermix in zygotes, indicating fusion of the parental mitochondrial membranes [20]. A conditional mutant, fzo1-1, exhibits rapid and extensive fragmentation of mitochondria already 10 min after shift to the nonpermissive temperature. When fzo1-1 cells are crossed at the nonpermissive temperature, mitochondrial networks fragment and fail to fuse. Fusion is also strongly reduced in matings between fzo1-1 and wild-type cells, suggesting that functional Fzo1p is required on mitochondria of both haploid parents [25]. Mice lacking either one of the mfn1 or mfn2 genes die early during embryonic development. Mfn2 mutant mice show a specific defect in placental development, which might be the primary reason for embryonic lethality. Interestingly, a placental defect was not observed in Mfn1 mutant mice despite expression of Mfn1 in the placenta. Both Mfn1- and Mfn2-deficient cells harbor fragmented mitochondria that are deficient in mitochondrial fusion in tissue culture cells [51].
3.6. Overexpression of Fzo proteins A role of mammalian mitofusins in determining mitochondrial shape was first proposed based on overexpression studies in tissue culture cells. Transfected HeLa or COS-7 cells highly overexpressing Mfn2 exhibit massive perinuclear clustering of mitochondria [47,52]. Ultrastructural examination of these cells by electron microscopy revealed spherical or ovoid mitochondria with increased diameter and swollen cristae. Intriguingly, the membranes of apposing mitochondria were in close contact without merging [47]. This suggests that fusion might be blocked at the stage of adherence or docking of the organelles, possibly by accumulation of unproductive fusion intermediates. 3.7. Structure – function studies Functional analysis of a number of site-specific mutants in different organisms shed light on structure – function relationships of Fzo-related proteins. The GTPase domain contains four conserved motifs, G1 –G4, which are also found in other GTPases, where they are required for GTP binding and hydrolysis [53]. Most mutations of conserved amino acid residues within these motifs result in a complete loss of function of Fzo proteins. GTPase mutants of Drosophila Fzo are male sterile and defective in Nebenkern formation [8]; GTPase mutants of yeast Fzo1p harbor fragmented mitochondria and lose their mitochondrial genome [25]; and GTPase mutants of human Mfn2 are unable to generate extended mitochondrial networks under conditions of compromised mitochondrial fission [52]. These observations suggest that GTP binding and/or hydrolysis by Fzo is essential for mitochondrial fusion. On the other hand, a functional GTPase is not required for mitochondrial clustering by overexpression of Mfn2 in mammalian cells [47,52]. In contrast, a role in mitochondrial clustering was suggested for coiled-coil regions because a truncation mutant lacking the C-terminal coiled-coil of human Mfn2 showed a strong reduction of clustering [52]. Furthermore, coiled-coil regions are apparently important for inter- or intramolecular interactions of Fzo proteins. A C-terminal Mfn2 fragment covering the transmembrane segments and the C-terminal domain is able to recruit an otherwise soluble N-terminal fragment lacking the transmembrane segments to mitochondria, and is able to induce clustering. Mitochondrial localization and clustering depend on the coiled-coil domains in both fragments [47]. Several studies have demonstrated that the transmembrane segments and surrounding regions are important for targeting of Fzo proteins to the mitochondrial outer membrane [47,50,52]. Moreover, the intermembrane space loop between the transmembrane regions of Fzo1p was shown to be important for the protein’s function in yeast. Insertion of a flexible linker between both transmembrane domains abolishes the contact of the mutant protein to the inner membrane and causes fragmentation of mitochondria [50].
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3.8. Models for Fzo function The observations described above can be integrated into a—yet rather hypothetical—model of Fzo function. It is conceivable that an early step of mitochondrial fusion requires docking of two organelles approaching each other. This step might involve intermolecular interactions between Fzo proteins on opposite mitochondria mediated by coiledcoil domains. In addition, inter- or intramolecular coiled-coil interactions of molecules of the same membrane might be important. It has been speculated that Fzo coiled-coils form a four helix bundle [47], as it is also seen in SNARE proteins [54]. If such a multiple helix bundle is assembled from molecules of opposite organelles, it could act directly as a fusogen of the outer membrane. A putative self-interaction (as pictured in Fig. 3) is only one out of several possible assembly states of Fzo. Moreover, it cannot be excluded that other domains of Fzo and/or other proteins are involved. The GTPase could play a regulatory role in modulating the activity of Fzo or other components of the fusion machinery. Alternatively, the GTPase could provide biomechanical energy that could be used directly to overcome the energy barrier of membrane fusion. In the latter case, Fzo GTPases would act as mechanoenzymes similar to dynamin-like proteins [55,56]. Finally, the intermembrane space loop connects the outer membrane fusion complex to components in the inner membrane or intermembrane space to coordinate fusion of the mitochondrial outer and inner membranes [50]. Although this mode of action is compatible with all available data, many aspects of Fzo function are yet rather speculative, and many important questions remain unanswered. For example, it has not been demonstrated that Fzolike proteins act directly as fusogens for the outer membrane. It is equally well possible that Fzo family members are key regulators acting on yet unknown proteins that mediate mixing of the lipid bilayers. It can be expected that uncovering the composition of the Fzo1p-containing 800kDa complex in the outer membrane [24] and identification of putative interaction partners in the inner membrane [50] will help to clarify these issues in the future.
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was first identified in a screen for genes required for mitochondrial genome maintenance, MGM [58]. Later it was demonstrated that mgm1 mutants harbor abnormal mitochondria [59,60]. Examination of temperature-sensitive mgm1 mutants identified fragmentation of mitochondrial reticuli as the primary phenotype associated with loss of Mgm1p function [57]. Intriguingly, mitochondrial fragmentation and loss of mitochondrial DNA in mgm1 mutants are suppressed by mutations of components of mitochondrial fission [57,61 – 63], and mitochondrial fusion is blocked in zygotes lacking Mgm1p [36,64]. These phenotypes are very similar to that observed in fzo1 mutant cells, suggesting that Mgm1p might also be required for mitochondrial fusion. Moreover, Mgm1p was found to physically interact with fusion factors in yeast, Fzo1p and Ugo1p [64]. However, these interactions might be only transient, and their mechanistic significance is not fully understood. The intramitochondrial localization of Mgm1p and its orthologs has been a matter of debate [57,60,65]. Recent results are in favor of a localization in the intermembrane space [36,64,66]. The precise role of Mgm1-like proteins in mitochondrial morphogenesis is not known. Suggested functions include reorganization of mitochondria following a change in carbon source [59], anchoring mitochondrial DNA to the inner membrane [67], local influence on the structural properties of mitochondrial membranes [60], fission and/or remodelling of the inner membrane [57,65], formation or stabilization of inner membrane cristae [57,65,66], and coordination of fusion of the mitochondrial membranes [64]. Thus, it is at present unclear whether the role of Mgm1-related proteins in mitochondrial fusion is direct or indirect. Elucidation of the molecular role of Mgm1p and its orthologues will be of considerable interest because OPA1, the human homolog of MGM1, has recently been identified as the gene responsible for dominant optical atrophy (DOA), the most common form of inherited optic neuropathy [68,69]. This disease results in a progressive loss of retinal ganglion cells that is associated with childhood and adult blindness and may represent the first case of a mitochondrial disease caused by an alteration of mitochondrial dynamics [70].
4. A role of dynamin-like proteins in mitochondrial fusion? 5. Additional lessons from yeast Dynamin is a mechanoenzyme that mediates the release of clathrin-coated vesicles from the plasma membrane during endocytosis. Recently, a number of proteins related to this large GTPase have been implicated in a variety of membrane trafficking events, including maintenance of mitochondrial morphology [38,55,56]. In particular, dynamin-like proteins assembling on the mitochondrial surface are thought to mediate mitochondrial division by severing the mitochondrial membranes [36,38 – 40]. A role in mitochondrial fusion has been proposed for the dynamin-like protein Mgm1p in yeast [57]. This component
Genetic screens in yeast have identified additional components that might play important roles in mitochondrial fusion. Ugo1p is a novel protein of the mitochondrial outer membrane. The phenotype of yeast cells lacking Ugo1p resembles that of fzo1 and mgm1 mutants. Dugo1 mutants harbor fragmented mitochondria, lose mitochondrial DNA and fail to fuse mitochondria in zygotes [62]. As Ugo1p does not localize with Fzo1p to sites of close contact between the mitochondrial outer and inner membranes, and as evidence for an interaction of Ugo1p and Fzo1p
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could not be obtained, it is likely that these two proteins do not permanently interact. Thus, they presumably mediate distinct steps in mitochondrial fusion [62]. A comprehensive genome-wide screen for yeast mutants with defective mitochondrial distribution and morphology recently uncovered a number of novel genes involved in mitochondrial morphogenesis. A deletion mutant library covering 4794 nonessential yeast genes was systematically screened with mitochondria-specific fluorescent probes. Ten previously uncharacterized genes were identified to be essential for establishment and maintenance of a wild type-like mitochondrial morphology [71]. Some of these novel mdm mutants displayed fragmented mitochondria, suggesting a defect in mitochondrial fusion. This class of mutants includes mdm30 and mdm37. Certain motifs within the Mdm30p and Mdm37p sequences point to a possible role of these proteins as regulators of mitochondrial fusion. The Mdm30p sequence contains an F-box motif which is commonly found in subunits of Skp1-Cdc53-F-box protein (SCF) ubiquitin ligases [72]. Cells lacking Mdm30p contain highly aggregated or fragmented mitochondria and fail to fuse mitochondria in zygotes. The protein level of Fzo1p is regulated by Mdm30p. Thus, Mdm30p controls mitochondrial shape by regulating the steady state level of Fzo1 and apparently links the ubiquitin/26S proteasome system to mitochondria [73]. Mdm37p shares homology with the rhomboid protein of Drosophila, which is involved in proteolytic activation of factors of the epidermal growth factor signalling pathway [74]. Recently, yeast Mdm37p (alternative name Pcp1p) was shown to be involved in proteolytic processing of the enzyme cytochrome c peroxidase in the intermembrane space [75]. It is tempting to speculate that Mdm37p is involved in proteolytic activation of mitochondrial fusion factors in a similar manner.
6. Perspectives Fusion of mitochondria requires faithful merging of four membranes that must be intimately coordinated with organellar fission processes. Given the complexity of this reaction, it can be expected that several important proteins contributing to this process remain to be discovered. These will likely include components of the fusion machinery in the mitochondrial inner membrane, regulatory proteins and maybe additional factors in the outer membrane and intermembrane space. Dissecting the biochemical roles of known and novel players of mitochondrial fusion will require the development of in vitro assays and reconstitution of individual steps of the fusion process. Detailed analysis of knock-out mutants in animal model systems will teach us more about the role of mitochondrial fusion in cell development, apoptosis, aging and physiology. Clearly, the elucidation of the cellular role and molecular mechanisms of
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