- Email: [email protected]
Regulation of mitochondrial fusion and division
Review
TRENDS in Cell Biology
Vol.17 No.11
Regulation of mitochondrial fusion and division Kara L. Cerveny1, Yasushi Tamura2, Zhongyan Zhang2, Robert E. Jensen2 and Hiromi Sesaki2 1 2
The Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK Department of Cell Biology, The Johns Hopkins University School of Medicine Baltimore, MD 21205, USA
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. Introduction Mitochondria acquire specialized shapes, undergo changes in number and intracellular distribution and reorganize their morphology and intricate double-membrane structures dramatically, often in response to the metabolic needs of the cell. A growing body of evidence indicates that mitochondrial fusion and fission have important roles in establishing, maintaining and remodeling mitochondria [1–3] (Figure 1). The mitochondria of many cell types appear to fuse and divide continuously in a highly regulated manner, such that their overall structure can change rapidly in response to different biological cues. Examples include elongation of mitochondrial tubules during differentiation of embryonic stem cells into cardiomyocytes [4], increased mitochondrial division during synapse formation in hippocampal neurons to recruit mitochondria into the neural protrusions [5] and fragmentation of mitochondria correlating with cytochrome c release during apoptosis [6,7]. Demonstrating the importance of mitochondrial dynamics in human health is the fact that defects in organelle fusion and fission lead to a variety of diseases. For example, mutations in mitochondrial-fusion components are associated with neurodegenerative diseases, such as Charcot-Marie-Tooth (CMT) disease type 2A (CMT2A) and the progressive blindness condition, dominant optic atrophy 1 (DOA1) [8,9]. Similarly, abnormalities in mitochondrial division are associated with CMT Corresponding author: Sesaki, H. ([email protected]). Available online 23 October 2007. www.sciencedirect.com
type 4A and defects in embryonic and postnatal development, including abnormal brain and retinal development. [10–12]. In the last decade, many studies have identified and characterized proteins responsible for organelle fusion and division [3,13] and have demonstrated that a balance of these two antagonistic activities controls mitochondrial morphology. Only now are we beginning to gain an appreciation of the molecular mechanisms that regulate these processes. Here, we highlight recent progress made towards understanding the mechanisms that control the frequency and location of mitochondrial fusion and fission. Regulation of mitochondrial fusion Mitochondria fuse using mechanisms distinct, or at least more complex, from those of other membrane-bound organelles [14]. This might reflect the endosymbiotic origin of these organelles, which have two membranes [an outer (OM) and an inner (IM)] that have distinct lipid and protein compositions. The fusion reaction must coordinate OM and IM events while maintaining the integrity of the two membranes. Like other membrane-fusion events, mitochondria are first tethered together before their OM and then IM bilayers mix. Two evolutionarily conserved large GTPases, Mitofusins (called Fzo1p in yeast) and OPA1 (named Mgm1p in yeast), have central roles in OM and IM fusion, respectively [2,15–21]. Studies using yeast proteins and isolated mitochondria or cultured mammalian cells have shown that these proteins participate in tethering and merging lipid bilayers [22–25]. Mitofusins are anchored in the mitochondrial OM by a bipartite transmembrane domain, such that both the N-terminal GTPase and carboxyl-terminal coiled-coil domains face the cytosol [26], whereas OPA1 (Mgm1p) proteins are found in the intermembrane space (IMS) where they associate with both the IM and OM [17]. Exciting new results demonstrate that these GTPases are also targets for the signals that control mitochondrial fusion. Later, we summarize how protein–protein interactions, post-translational modifications, protein turnover and the lipid environment contribute to the regulation of organelle fusion (Table 1). Hetero-oligomeric complexes of Mfn1 and 2 Mitofusins are related distantly to dynamin, the large GTPase required for vesicle endocytosis, and, like dynamin, these proteins oligomerize and hydrolyze GTP to
0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.08.006
Review
564
TRENDS in Cell Biology Vol.17 No.11
Figure 1. Fusion and division regulate mitochondrial shape antagonistically. Morphology of yeast mitochondria of wild-type cells and mutants defective for fusion, division or both are shown. (a). Wild-type cells contain tubular mitochondria with occasional branches. (b). Fusion mutants contain fragmented mitochondria owing to ongoing division, whereas (c) division mutants form a single mitochondrial consisting of interconnected tubules. (d). In cells without fusion and division activities, tubular mitochondria, similar to those seen in the wild type, are restored. Images were modified from Sesaki and Jensen, with permission [84].
catalyze the membrane rearrangements that lead to mitochondrial fusion. In addition, recent in vitro membranefusion studies suggest that the yeast mitofusin, Fzo1p, forms complexes that act in trans to fuse mitochondria [24]. Our understanding of mammalian mitochondrial fusion is complicated by the existence of two ubiquitously expressed vertebrate-specific mitofusin isoforms, Mfn1 and Mfn2, which can assemble into homo- and heterooligomers [27]. Although some experiments suggest that these proteins are redundant functionally [27], other experiments indicate that Mfn1 and Mfn2 have specialized functions. For example, Mfn1 exhibits higher GTPase activity and tighter mitochondrial tethering ability than Mfn2 [23]. In addition, Mfn1, but not Mfn2, is required for OPA1 function [28], whereas mutations in Mfn2 cause CMT2A [29].
Detailed analysis of the mutations that cause CMT2A has provided insight into the role of Mfn1–Mfn2 interactions. CMT2A is a progressive neurodegenerative disease characterized by the deterioration of long sensory and motor neuron axons. Characterization of multiple CMT2Aassociated Mfn2 alleles, which are concentrated near and in the GTPase domain, but are also found in the C-terminal oligomerization domain [30], illustrates that many mutant forms of Mfn2 can be rescued although interaction with Mfn1, but not Mfn2. Emphasizing the vital role that hetero-oligomeric complexes of Mfn1 and Mfn2 have during mitochondrial fusion, this functional complementation occurs in trans between Mfn1 and Mfn2 on opposite mitochondria. Furthermore, the specific peripheral neuropathies characteristic of CMT2A might be explained, at least in part, by the ratio of Mfn1 and Mfn2 in different cell types. For instance, cells with high levels of Mfn1, such as cardiomyocytes [31], might be protected from the diseasecausing Mfn2 protein because Mfn1 can form a functional complex with the mutant molecule [30]. However, cells that have lower levels of Mfn1 or that require higher levels of mitochondrial fusion, such as the long peripheral neurons most affected in CMT2A, might be particularly vulnerable to point mutations in Mfn2. With reduced mitochondrial fusion (and therefore a reduction in the distribution of mitochondria), these nerve cells might disperse ATP at the pre- and post-synaptic terminals less efficiently. Studies of a variety of neurodegenerative diseases indicate that appropriately controlled mitochondrial dynamics are crucial for the long-term maintenance of the nervous system [32]. Degradation of Fzo1p (Mfn) Modulating the amount of Mfn protein regulates the extent of mitochondrial fusion. For example, in wild-type yeast
Table 1. Regulation of mitochondrial dynamics Function Fusion
Organism Mammals
Protein Mitofusin 2
OPA1
Yeast
Fzo1p Mgm1p
Division
Mammals
Yeast
www.sciencedirect.com
Drp1
Fis1 Dnm1p
Regulator Bax and Bak Bcl-xL Mitofusin 1 MARCH-V/MITOL MIB Stomatin-like protein 2 PARL Paraplegin Membrane potential Mdm30p Proteasome Pcp1p PAM complex ATP Ups1p cyclin B-CDK cAMP-dependent kinase Sumo-1 SENP5 Bax and Bak Fis1 MARCH-V/MITOL MARCH-V/MITOL Fis1p Mdv1p Caf4p Num1p
Activity Assembly of Mfn2-containing complexes Unknown Complementation of mutant forms of Mfn2 in CMT 2A Possible ubiquitination and degradation Unknown Unkonwn Proteolytic processing Proteolytic processing Proteolytic processing Degradation during growth Degradation during mating Proteolytic processing Proteolytic processing Proteolytic processing Proteolytic processing Phosphorylation during cell cycle Inhibition of assembly and GTPase activitiy by phosphorylation Sumoylaiton Removal of SUMO Mitochondrial association of Drp1 Mitochondrial association of Drp1 Ubiquitination and degradation Ubiquitination and degradation Mitochondrial association of Dnm1p Mitochondrial association of Dnm1p Mitochondrial association of Dnm1p Intracellular dynamics of Dnm1p
Review
TRENDS in Cell Biology
cells, the yeast Mfn homologue, Fzo1p, is turned-over and degraded rapidly by at least two distinct mechanisms [26,33]. During vegetative growth, Fzo1p stability is influenced by the OM F-box-containing protein, Mdm30p. F-boxes are present in subunits of a subset of E3 ubiquitin ligases and are often required for the ubiquitin-conjugating activity of these proteins. Cells expressing a F-box-mutant version of Mdm30p accumulate Fzo1p, contain fragmented mitochondria and exhibit reduced mitochondrial fusion [26], whereas a genome-wide analysis showed that Fzo1p is ubiquitinated [34]. Surprisingly, constitutive Fzo1p turn-over does not require known ubiquitin or proteasome machinery, suggesting that Mdm30p-mediated degradation can occur independently of the proteasome [35,36]. Perhaps yeast cells use autophagy or mitochondrial proteases, such as the m-AAA proteases, to degrade Fzo1p. In contrast to vegetatively growing cells, yeast cells treated with mating pheromone for extended periods of time contain fragmented mitochondria and Fzo1p is degraded by the ubiquitin and proteasome pathway. Interestingly, this process does not appear to involve Mdm30p and it can be blocked when proteasome activity is inhibited pharmacologically [33]. Perhaps proteasome-mediated degradation of yeast Fzo1p favors organelle fragmentation during a-factor-induced cell death, which appears similar to the fragmentation of mitochondria seen in mammalian apoptosis [6,7]. To date, no studies have examined the half-life of Mfns or determined if Mfns are ubiquitinylated. Therefore, it will be interesting to test whether the same rules that govern Fzo1p turn-over impact the function of Mfns in healthy and apoptotic mammalian cells. Assembly of mitofusin (Mfn) complexes by Bcl-2 proteins Mitochondrial fusion and division are regulated during apoptosis. On apoptotic stimulation, the balance between fusion and division is disrupted and the mitochondria fragment [6,7]. Two pro-apoptotic Bcl-2 family members, Bax and Bak, have been implicated in mitochondrial dynamics during programmed cell death and Bax colocalizes with both fusion and fission proteins [37]. Interestingly, Bax and Bak also affect mitochondrial fusion in nonapoptotic cells by regulating the assembly of Mfn2 into high molecular-weight complexes [38]. In the absence of Bak and Bax, Mfn2 complexes become smaller and Mfn2 localization changes from discrete puncta on the organelle surface to a uniform distribution along the mitochondria. Similar, but not identical to Mfn2 deficient cells, Bax/Bak double knockout cells fail to fuse their mitochondria efficiently; as a result, they contain many short mitochondrial tubules. In addition to a role for pro-apoptotic proteins in mitochondrial fusion, two anti-apoptotic Bcl-2 family members – Bcl-xL and Caenorhabditis elegans CED-9 – also promote mitochondrial fusion in mammalian cells by interacting physically with Mfn2 [39]. These findings reveal unexpected non-apoptotic functions for both pro- and antideath Bcl-2 family members and hint that these proteins might exert their reciprocal control over programmed cell death by regulating the assembly of mitochondrial fusion proteins. www.sciencedirect.com
Vol.17 No.11
565
Possible regulation by novel Mfn-binding proteins Recent biochemical studies using mammalian cells have identified two additional Mfn partners, mitofusin-binding protein (MIB) and Stoml2. MIB was isolated as a cytosolic Mfn1-binding protein that associates with the mitochondrial OM and is similar to vesticle amine transferase 1 (VAT-1), an ATPase found in synaptic vesicles predominantly [40]. MIB is a member of the medium-chain dehydrogenase and reductase protein superfamily and it contains a conserved coenzyme-binding domain that might bind and hydrolyze nucleotides. Overexpression of MIB induces mitochondrial fragmentation whereas its knockdown results in a large extension of mitochondrial networks [40]. These phenotypes are consistent with MIB acting either as an activator of mitochondrial division or as an inhibitor of mitochondrial fusion. Consistent with this second possibility, MIB binds the fusion proteins Mfn1 and Mfn2 but not the division protein Drp1. Perhaps MIB uses its nucleotide-binding region to recognize Mfn1–GTP to prevent unproductive nucleotide hydrolysis by Mfn1 until it is fusion complex is assembled or positioned at fusion sites appropriately [40]. Stoml2 is a stomatin-like protein localized to the mitochondrial IMS where it binds Mfn2 [41]. Although knockdown of Stoml2 reduces mitochondrial-membrane potential, this treatment does not alter mitochondrial morphology dramatically. Currently, the role of Stoml2 in mitochondrial dynamics is unknown; however, its prohibitin-like domain might provide some clues to the function of Stoml2. In yeast mitochondria, prohibitins form large oligomeric complexes that perform a variety of functions, including chaperoning mitochondrial (mt)DNA-encoded proteins so that they assemble and insert into the membrane properly. Therefore, Stoml2 might chaperone Mfn2 to help assemble fusion-competent protein complexes. Proteolytic processing of Mgm1p (OPA1) Like the OM Mfn proteins, the machinery required for IM fusion is subjected to modifications that control mitochondrial fusion. Yeast Mgm1p and its mammalian counterpart OPA1 are required for mitochondrial fusion and IM cristae morphology [2,15–17,25]. Mgm1p is processed proteolytically into long (l-Mgm1p) and short forms (s-Mgm1p) inside mitochondria [42–44]. l-Mgm1p is integrated into the IM through its N-terminal transmembrane segment and its mitochondria-targeting presequence is cleaved by the matrix-processing protease. By contrast, s-Mgm1p is processed proteolytically in the IM and then released into the IMS. These two forms are present in approximately equal amounts and seem to perform overlapping as well as distinct roles in maintaining mtDNA, remodeling cristae and fusing mitochondria. Recently, experiments in yeast elucidated the mechanisms that produce l-Mgm1p and s-Mgm1p. s-Mgm1 is generated when Mgm1p is cleaved by the rhomboid-related protease Pcp1p in the IM. The levels of Pcp1p, in addition to the concentration of matrix ATP, regulate the production of s-Mgm1p [43,45]. Cells might use this mechanism to monitor the level of ATP and use this functional readout to inhibit the fusion of defective mitochondria. Ups1p, a protein of evolutionary and lymphoid interest (PRELI), and an evolutionarily conserved
566
Review
TRENDS in Cell Biology Vol.17 No.11
IMS protein, regulates the relative amounts of long and short forms of Mgm1p by controlling the insertion of Mgm1p’s Pcp1p-sensitive rhomboid-cleavage site into the IM [46]. Similar events control the processing of OPA1 in mammals. Similar to Mgm1p processing, OPA1 cleavage reflects the energy level in mitochondria and can be regulated by the membrane potential across the IM [47]. However, the identity of the protease that cleaves OPA1 is controversial. The mammalian homologue of yeast Pcp1p, called presenilin-associated rhomboid-like protein (PARL), has been implicated in OPA1 proteolysis. PARL does not appear to regulate the function of OPA1 in mitochondrial fusion, although it does influence the ability of Opa1p to remodel cristae [48,49]. Supporting its role in mitochondrial morphology, phosphorylation of PARL affects mitochondrial shape; however, it remains to be determined whether the phosphorylation of PARL modulates Opa1 cleavage [50]. At least three other proteins have been associated with OPA1 processing. The two m-AAA proteases AFG3L2 [51] and Paraplegin [52] and the i-AAA protease Yme1 [53,54] process OPA1 and it is likely that these three AAA proteases might combine in various ratios to insure OPA1 cleavage under a variety of cellular conditions [51]. Furthermore, because mammalian cells contain at least eight different splice variants of OPA1 [55], it is likely that many versions of OPA1 contribute to mitochondrial remodeling. Clearly, further investigations into the proteins and mechanisms that contribute to the functions of OPA1 are warranted. Lipid modification by phospholipase D The lipid composition of the mitochondrial membrane bilayers can influence membrane remodeling. Indeed, some lipids, such as phosphatidic acid (PA), are particularly fusogenic and seem to contribute to stereotypical changes in membrane curvature that thermodynamically favor deformation and bilayer mixing [56]. Choi et al. [57] identified a mitochondria-targeted member of the phospholipase D (PLD) protein family that is required for mitochondrial fusion. This highly divergent lipase, named MitoPLD, catalyzes the synthesis of PA from the mitochondria-enriched lipid, cardiolipin. The catalytic site of MitoPLD is located on the cytosolic surface of the OM. Because the substrate for this protein, cardiolipin, is located mainly in the IM, MitoPLD might catalyze fusion at contact sites, places where the IM and OM are closely apposed, which are thought to provide conduits for lipid and protein transfer from one membrane to the other [17,58–60]. Many questions about how MitoPLD functions remain. For instance, does its hydrolysis of cardiolipin activate the membrane-anchored fusion proteins or does it simply alter membrane curvature to favor lipid mixing? The isolation and characterization of this interesting protein has provided the first step towards understanding how the local lipid environment can facilitate mitochondrial dynamics. Regulation of mitochondrial division Similar to mitochondrial fusion, mitochondrial division requires a dynamin-related GTPase, called Dnm1p in www.sciencedirect.com
yeast and Drp1 in mammals [61–65]. In contrast to the integral membrane-fusion proteins, these proteins shuttle between the cytosol and the surface of mitochondria. Dnm1p (Drp1) is recruited to the mitochondrial surface and assembles into oligomeric complexes, which are thought to wrap around mitochondria surface by a variety of receptors – Mdv1, Caf4, and Fis – and then assembles into oligomeric complexes [66–69]. As these complexes assemble, it has been proposed that they wrap around the mitochondrial tubules like spirals that constrict and eventually divide mitochondria [70]. Below we summarize recent findings that have uncovered new regulatory mechanisms that control mitochondrial fission in response to a variety of cellular events, including cell division, metabolic flux and cell differentiation. Most of these processes seem to regulate the localization, dynamics and activity of Dnm1p (Drp1) (Table 1). Phosphorylation of Drp1 Mitochondrial division is coordinated with the cell cycle in higher eukaryotes. Recent experiments using cultured human cells showed elegantly that mitochondrial scission is induced at the onset of mitosis, leading to partial fragmentation of mitochondria [71]. Revealing a direct link between the cell-cycle and the mitochondrial-division machinery, this burst of mitochondrial division is correlated with the cyclinB–cyclin-dependant kinase (CDK1dependent) phosphorylation of Drp1. This is the first demonstration that the addition of a phosphate moiety to Drp1 regulates its activity and in vitro assays using purified proteins coupled with cell-culture experiments indicate that the most potent mitotic phosphorylation event occurs on a serine residue in the carboxyl-terminal GTPase-effecter domain (GED) of Drp1. Another recent study showed that cyclic-AMP-kinase-dependent phosphorylation of a different serine in the GED can decrease the GTPase activity of Drp1 by inhibiting the intramolecular interactions known to increase the GTPase activity of Drp1 [72]. Although these initial studies indicate that Drp1 phosphorylation can modulate the frequency of mitochondrial division, it remains to be determined if fissioncompetent Drp1 is always phosphorylated or if this is a mechanism exploited only during the cell cycle. In addition, it will be useful to learn if the phosphorylation state of Dnm1p influences mitochondrial division in yeast, especially during mitosis. Ubiquitinylation of Drp1 Ubiquitinylation regulates mitochondrial division. Experiments using yeast mitochondria have raised the possibility that ubiquitin conjugation to mitochondria controls a variety of organelle processes, including mitochondrial dynamics and inheritance [73]. The most convincing evidence to date that ubiquitinylation controls mitochondrial division comes from three independent investigations of the membrane-associated RING-CH (MARCH-V (MITOL)) E3 ubiquitin ligase in mammalian cells. This OM-anchored protein is capable of binding and modifying Drp1 and its OM anchor, Fis1 [74–76]. Although the data from these studies support conflicting functions for MARCH-V – either as an activator or an inhibitor of fission – it is clear
Review
TRENDS in Cell Biology
that binding between Drp1 and MARCH-V influences mitochondrial dynamics. The most recent work [76] suggests that MARCH-V ubiquitinylates Drp1 but does not control the stability of Drp1. Instead, Drp1 ubiquitinylation seems to regulate the kinetics of Drp1 binding to the mitochondrial surface. In this instance, ubiquitin conjugation might regulate the subcellular trafficking, assembly of Drp1 and influence the rate of mitochondrial division [76]. Much remains to be learned about the stability and trafficking of Drp1. In particular, it will be interesting to ascertain if particular signals, such as cellular stresses or cell-cycle progression, influence the frequency and amount of ubiquitinylated Drp1 and if the activity of the yeast cousin of Drp1, Dnm1p, is modified by ubiquitin. Sumoylation of Drp1 The small ubiquitin-like modifier (SUMO) is highly conserved from yeast to humans and can be conjugated to a wide variety of proteins in a manner similar to ubiquitin. Unlike ubiquitinylation, which often leads to degradation of substrates, sumoylation usually alters the subcellular localization of target proteins or protects them from ubiquitin-mediated destruction. Drp1 pull-down experiments identified SUMO-1 and its conjugating enzyme Ubc9 as Drp1-binding partners [77]. Confirming that these two proteins form a functional complex that binds and modifies Drp1, sumoylated Drp1 has been isolated from mammalian cells [77]. Consistent with the idea that sumoylated Drp1 promotes mitochondrial division, overexpressing SUMO1 or silencing sentrin/SUMO-specific protease (SENP5), the protease that removes SUMO from Drp1, yields cells with many small mitochondria [77,78]. Interestingly, SENP5 is also a key player in cell-cycle progression [79] and, similar to the cyclinB–CDK1 dependent phosphorylation of Drp1, the interactions among Drp1, SUMO and SENP5 might help
Vol.17 No.11
567
to coordinate mitochondrial division and the cell cycle to insure even segregation of mitochondria. Regulation of Drp1 association with mitochondria by Bax and Bak Sumoylation of Drp1 also appears to influence the integration of pro-apoptotic signals and mitochondrial division. Recent experiments illustrate that normal cycling of Drp1 on and off the organelle surface is arrested during apoptosis, such that Drp1 accumulates on the mitochondria [80]. In this scenario, Drp1 is recruited to the mitochondrial membrane independent of its well characterized membrane anchor, Fisp1. In addition, Drp1 becomes sumoylated in a Bax- and Bak-dependent manner, suggesting that these pro-apoptotic proteins enhance the mitochondrial association of Drp1 through sumoylation. These events immediately precede apoptosis, which raises the possibility that sumoylation might alter the activity of Drp1 as a result of apoptotic signals thereby contributing to mitochondrial fragmentation during programmed cell death. Regulation of Dnm1p dynamics by Num1p In addition to the mechanisms that modulate mitochondrial division in mammalian cells, many early studies exploited the genetic potential of yeast to understand how mitochondria divide [3,81]. The latest of these studies, which searched for high-copy suppressors of mutant Dnm1p (the yeast orthologue of Drp1), found that the cortically localized multidomain Num1 protein affects mitochondrial division. Many previous studies demonstrated that Num1p controls nuclear migration and inheritance through its association with components of both microtubule and actin cytoskeletons [82]. In addition to this well established role for Num1p, it also participates in the division and inheritance of mitochondria. Reminiscent of the
Figure 2. Regulation of mitochondrial fusion and division. Schematic of organelle dynamics highlights many of the processes that regulate mitochondrial dynamics and illustrates the consequences of unbalanced fusion or division. Defects in mitochondrial fusion (unbalanced division) lead to a variety of pathologies, including CharcotMarie-Tooth Disease type 2A (CMT type 2A) and dominant optic atrophy (DOA). In addition, excessive mitochondrial fragmentation accompanies apoptosis. Defects in mitochondrial division (unbalanced fusion) cause defects in neural development and Charcot-Marie-Tooth Disease type 4 (CMT type 4). www.sciencedirect.com
568
Review
TRENDS in Cell Biology Vol.17 No.11
control that MARCH-V and SUMO-1 exert over Drp1 trafficking, Num1p appears to regulate mitochondrial division by influencing the dynamic interactions of Dnm1p with mitochondria. Not only is Num1p important for mitochondrial division but it also cooperates with Dnm1p to insure mitochondrial inheritance during mitosis [83]. Given the intimate association of Num1p with the cytoskeleton, its function in nuclear migration and its physical interaction with Dnm1p, Num1p is positioned perfectly to coordinate organelle division and inheritance. Whether similar proteins capable of linking organelle division with inheritance exist in mammalian cells awaits further inquiry. Conclusions and perspectives The dynamic equilibrium between fusion and division enables rapid and robust changes in mitochondrial structure and function by stimulating one activity and/or repressing the other (Figure 2). Reinforcing the importance of the antagonistic relationship between mitochondrial fusion and division, some of the factors that influence mitochondrial fusion, such as Bcl-2 proteins and ubiquitin, also have the ability to modulate the opposing process of division. Moreover, identification and study of the mechanisms that integrate cellular processes, such as cell division and apoptosis, with mitochondrial remodeling, have yielded glimpses into how a cell’s physiological context can influence mitochondrial morphology and function. Whether specific cell types have evolved unique mechanisms to adapt their mitochondrial morphology in response to intrinsic and extrinsic signals remains an open question. Recent publications have reported on many of the signals that regulate mitochondrial dynamics, including the diverse processes of phosphorylation, ubiquitinylation, proteolytic processing, protein trafficking, lipid remodeling and assembly of functional protein complexes, however, we know very little about how all of these signals are connected. Continued investigation into the proteins and lipids that regulate mitochondrial dynamics will contribute new insight to our understanding of how mitochondrial fusion and division are coordinated with the developmental and metabolic needs of eukaryotic organisms. Acknowledgements This work was supported by Damon Runyon Cancer Research Foundation (DRG1877–05) (K.L.C), Uehara Memorial Foundation (Y.T.), National Institutes of Health (R.E.J.), Johns Hopkins University and American Heart Association (H.S.).
References 1 Osteryoung, K.W. and Nunnari, J. (2003) The division of endosymbiotic organelles. Science 302, 1698–1704 2 Griparic, L. et al. (2004) Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792–18798 3 Okamoto, K. and Shaw, J.M. (2005) Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu. Rev. Genet. 39, 503–536 4 Chung, S. et al. (2007) Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat. Clin. Pract. Cardiovasc. Med. 4 (Suppl. 1), S60–S67 5 Li, Z. et al. (2004) The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873–887 6 Cereghetti, G.M. and Scorrano, L. (2006) The many shapes of mitochondrial death. Oncogene 25, 4717–4724 www.sciencedirect.com
7 Youle, R.J. and Karbowski, M. (2005) Mitochondrial fission in apoptosis. Nat. Rev. Mol. Cell Biol. 6, 657–663 8 Olichon, A. et al. (2006) Mitochondrial dynamics and disease, OPA1. Biochim. Biophys. Acta 1763, 500–509 9 Chan, D.C. (2006) Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241–1252 10 Waterham, H.R. et al. (2007) A lethal defect of mitochondrial and peroxisomal fission. N. Engl. J. Med. 356, 1736–1741 11 Cuesta, A. et al. (2002) The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal CharcotMarie-Tooth type 4A disease. Nat. Genet. 30, 22–25 12 Niemann, A. et al. (2005) Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease. J. Cell Biol. 160, 1067–1078 13 Chan, D.C. (2006) Mitochondrial fusion and fission in mammals. Annu. Rev. Cell Dev. Biol. 22, 79–99 14 Meeusen, S.L. and Nunnari, J. (2005) How mitochondria fuse. Curr. Opin. Cell Biol. 17, 389–394 15 Olichon, A. et al. (2003) Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. J. Biol. Chem. 278, 7743–7746 16 Wong, E.D. et al. (2000) The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J. Cell Biol. 151, 341–352 17 Sesaki, H. et al. (2003) Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane. Mol. Biol. Cell 14, 2342–2356 18 Santel, A. and Fuller, M.T. (2001) Control of mitochondrial morphology by a human mitofusin. J. Cell Sci. 114, 867–874 19 Hermann, G.J. et al. (1998) Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p. J. Cell Biol. 143, 359–373 20 Rapaport, D. et al. (1998) Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae. J. Biol. Chem. 273, 20150–20155 21 Hales, K.G. and Fuller, M.T. (1997) Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90, 121–129 22 Koshiba, T. et al. (2004) Structural basis of mitochondrial tethering by mitofusin complexes. Science 305, 858–862 23 Ishihara, N. et al. (2004) Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity. J. Cell Sci. 117, 6535–6546 24 Meeusen, S. et al. (2004) Mitochondrial fusion intermediates revealed in vitro. Science 305, 1747–1752 25 Meeusen, S. et al. (2006) Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Cell 127, 383–395 26 Fritz, S. et al. (2003) Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast. Mol. Biol. Cell 14, 2303–2313 27 Chen, H. et al. (2003) Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J. Cell Biol. 160, 189–200 28 Cipolat, S. et al. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U. S. A. 101, 15927–15932 29 Zuchner, S. et al. (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat. Genet. 36, 449–451 30 Detmer, S.A. and Chan, D.C. (2007) Complementation between mouse Mfn1 and Mfn2 protects mitochondrial fusion defects caused by CMT2A disease mutations. J. Cell Biol. 176, 405–414 31 Santel, A. et al. (2003) Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells. J. Cell Sci. 116, 2763–2774 32 Mandemakers, W. et al. (2007) A cell biological perspective on mitochondrial dysfunction in Parkinson disease and other neurodegenerative diseases. J. Cell Sci. 120, 1707–1716 33 Neutzner, A. and Youle, R.J. (2005) Instability of the mitofusin Fzo1 regulates mitochondrial morphology during the mating response of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 280, 18598–18603 34 Hitchcock, A.L. et al. (2003) A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum
Review
35
36
37
38 39 40
41
42
43 44
45
46
47
48 49
50
51
52 53
54 55 56 57 58
TRENDS in Cell Biology
degradation machinery. Proc. Natl. Acad. Sci. U. S. A. 100, 12735– 12740 Durr, M. et al. (2006) Nonredundant roles of mitochondria-associated F-box proteins Mfb1 and Mdm30 in maintenance of mitochondrial morphology in yeast. Mol. Biol. Cell 17, 3745–3755 Escobar-Henriques, M. et al. (2006) Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1. J. Cell Biol. 173, 645–650 Karbowski, M. et al. (2002) Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J. Cell Biol. 159, 931–938 Karbowski, M. et al. (2006) Role of Bax and Bak in mitochondrial morphogenesis. Nature 443, 658–662 Delivani, P. et al. (2006) Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics. Mol. Cell 21, 761–773 Eura, Y. et al. (2006) Identification of a novel protein that regulates mitochondrial fusion by modulating mitofusin (Mfn) protein function. J. Cell Sci. 119, 4913–4925 Hajek, P. et al. (2007) Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2. J. Biol. Chem. 282, 5670–5681 Sesaki, H. et al. (2003) Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependentmanner, but remain competent for mitochondrial fusion. Biochem. Biophys. Res. Commun. 308, 276–283 McQuibban, G.A. et al. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541 Herlan, M. et al. (2003) Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J. Biol. Chem. 278, 27781–27788 Herlan, M. et al. (2004) Alternative topogenesis of Mgm1 and mitochondrial morphology depend on ATP and a functional import motor. J. Cell Biol. 165, 167–173 Sesaki, H. et al. (2006) Ups1p, a conserved intermembrane space protein, regulates mitochondrial shape and alternative topogenesis of Mgm1p. J. Cell Biol. 173, 651–658 Duvezin-Caubet, S. et al. (2006) Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology. J. Biol. Chem. 281, 37972–37979 Frezza, C. et al. (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126, 177–189 Cipolat, S. et al. (2006) Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126, 163–175 Jeyaraju, D.V. et al. (2006) Phosphorylation and cleavage of presenilinassociated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology. Proc. Natl. Acad. Sci. U. S. A. 103, 18562–18567 Duvezin-Caubet, S. et al. (2007) OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria. Mol. Biol. Cell 18, 3582–3590 Ishihara, N. et al. (2006) Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. EMBO J. 25, 2966–2977 Song, Z. et al. (2007) OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L. J. Cell Biol. 178, 749–755 Griparic, L. et al. (2007) Regulation of the mitochondrial dynaminrelated protein Opa1 by proteolytic cleavage. J. Cell Biol. 178, 757–764 Delettre, C. et al. (2002) OPA1 (Kjer type) dominant optic atrophy: a novel mitochondrial disease. Mol. Genet. Metab. 75, 97–107 Chernomordik, L.V. et al. (2006) Membranes of the world unite! J. Cell Biol. 175, 201–207 Choi, S.Y. et al. (2006) A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis. Nat. Cell Biol. 8, 1255–1262 Fritz, S. et al. (2001) Connection of the mitochondrial outer and inner membranes byFzo1 is critical for organellar fusion. J. Cell Biol. 152, 683–692
www.sciencedirect.com
Vol.17 No.11
569
59 Sesaki, H. and Jensen, R.E. (2004) Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion. J. Biol. Chem. 279, 28298–28303 60 Wong, E.D. et al. (2003) The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion. J. Cell Biol. 160, 303–311 61 Labrousse, A.M. et al. (1999) C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol. Cell 4, 815–826 62 Smirnova, E. et al. (2001) Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol. Biol. Cell 12, 2245– 2256 63 Pitts, K.R. et al. (2004) Mitochondria-specific function of the dynamin family protein DLP1 is mediated by its C-terminal domains. J. Biol. Chem. 279, 50286–50294 64 Sesaki, H. and Jensen, R.E. (1999) Division versus fusion: Dnm1p and Fzo1 pantagonistically regulate mitochondrial shape. J. Cell Biol. 147, 699–706 65 Bleazard, W. et al. (1999) The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1, 298–304 66 Griffin, E.E. et al. (2005) The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria. J. Cell Biol. 170, 237–248 67 Cerveny, K.L. et al. (2001) Division of mitochondria requires a novel DNM1interacting protein, Net2p. Mol. Biol. Cell 12, 309–312 68 Tieu, Q. and Nunnari, J. (2000) Mdv1p Is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division. J. Cell Biol. 151, 353–366 69 Mozdy, A.D. et al. (2000) Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p. J. Cell Biol. 151, 367–380 70 Ingerman, E. et al. (2005) Dnm1 forms spirals that are structurally tailored to fit mitochondria. J. Cell Biol. 170, 1021–1027 71 Taguchi, N. et al. (2007) Mitotic phosphorylation of dynamin-related GTPase Drp1participates in mitochondrial fission. J. Biol. Chem. 282, 11521–11529 72 Chang, C.R. and Blackstone, C. (2007) Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology. J. Biol. Chem. 282, 21583–21587 73 Escobar-Henriques, M. and Langer, T. (2006) Mitochondrial shaping cuts. Biochim. Biophys. Acta 1763, 422–429 74 Nakamura, N. et al. (2006) MARCH-V is a novel mitofusin 2- and Drp1binding protein able to change mitochondrial morphology. EMBO Rep. 7, 1019–1022 75 Yonashiro, R. et al. (2006) A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J. 25, 3618–3626 76 Karbowski, M. et al. (2007) The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division. J. Cell Biol. 178, 71–84 77 Harder, Z. et al. (2004) Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission. Curr. Biol. 14, 340–345 78 Zunino, R. et al. (2007) The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. J. Cell Sci. 120, 1178–1188 79 Di Bacco, A. and Gill, G. (2006) SUMO-specific proteases and the cell cycle. An essential role for SENP5 in cell proliferation. Cell Cycle 5, 2310–2313 80 Wasiak, S. et al. (2007) Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death. J. Cell Biol. 177, 439–450 81 Jensen, R.E. et al. (2000) Yeast mitochondrial dynamics: fusion, division, segregation, and shape. Microsc. Res. Tech. 51, 573–583 82 Bloom, K. (2001) Nuclear migration: cortical anchors for cytoplasmic dynein. Curr. Biol. 11, R326–R329 83 Cerveny, K.L. et al. (2007) Yeast mitochondrial division and distribution require the cortical num1 protein. Dev. Cell 12, 363–375 84 Sesaki, H. and Jensen, R.E. (1999) Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J. Cell Biol. 147, 699–706
TRENDS in Cell Biology
Vol.17 No.11
Regulation of mitochondrial fusion and division Kara L. Cerveny1, Yasushi Tamura2, Zhongyan Zhang2, Robert E. Jensen2 and Hiromi Sesaki2 1 2
The Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK Department of Cell Biology, The Johns Hopkins University School of Medicine Baltimore, MD 21205, USA
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. Introduction Mitochondria acquire specialized shapes, undergo changes in number and intracellular distribution and reorganize their morphology and intricate double-membrane structures dramatically, often in response to the metabolic needs of the cell. A growing body of evidence indicates that mitochondrial fusion and fission have important roles in establishing, maintaining and remodeling mitochondria [1–3] (Figure 1). The mitochondria of many cell types appear to fuse and divide continuously in a highly regulated manner, such that their overall structure can change rapidly in response to different biological cues. Examples include elongation of mitochondrial tubules during differentiation of embryonic stem cells into cardiomyocytes [4], increased mitochondrial division during synapse formation in hippocampal neurons to recruit mitochondria into the neural protrusions [5] and fragmentation of mitochondria correlating with cytochrome c release during apoptosis [6,7]. Demonstrating the importance of mitochondrial dynamics in human health is the fact that defects in organelle fusion and fission lead to a variety of diseases. For example, mutations in mitochondrial-fusion components are associated with neurodegenerative diseases, such as Charcot-Marie-Tooth (CMT) disease type 2A (CMT2A) and the progressive blindness condition, dominant optic atrophy 1 (DOA1) [8,9]. Similarly, abnormalities in mitochondrial division are associated with CMT Corresponding author: Sesaki, H. ([email protected]). Available online 23 October 2007. www.sciencedirect.com
type 4A and defects in embryonic and postnatal development, including abnormal brain and retinal development. [10–12]. In the last decade, many studies have identified and characterized proteins responsible for organelle fusion and division [3,13] and have demonstrated that a balance of these two antagonistic activities controls mitochondrial morphology. Only now are we beginning to gain an appreciation of the molecular mechanisms that regulate these processes. Here, we highlight recent progress made towards understanding the mechanisms that control the frequency and location of mitochondrial fusion and fission. Regulation of mitochondrial fusion Mitochondria fuse using mechanisms distinct, or at least more complex, from those of other membrane-bound organelles [14]. This might reflect the endosymbiotic origin of these organelles, which have two membranes [an outer (OM) and an inner (IM)] that have distinct lipid and protein compositions. The fusion reaction must coordinate OM and IM events while maintaining the integrity of the two membranes. Like other membrane-fusion events, mitochondria are first tethered together before their OM and then IM bilayers mix. Two evolutionarily conserved large GTPases, Mitofusins (called Fzo1p in yeast) and OPA1 (named Mgm1p in yeast), have central roles in OM and IM fusion, respectively [2,15–21]. Studies using yeast proteins and isolated mitochondria or cultured mammalian cells have shown that these proteins participate in tethering and merging lipid bilayers [22–25]. Mitofusins are anchored in the mitochondrial OM by a bipartite transmembrane domain, such that both the N-terminal GTPase and carboxyl-terminal coiled-coil domains face the cytosol [26], whereas OPA1 (Mgm1p) proteins are found in the intermembrane space (IMS) where they associate with both the IM and OM [17]. Exciting new results demonstrate that these GTPases are also targets for the signals that control mitochondrial fusion. Later, we summarize how protein–protein interactions, post-translational modifications, protein turnover and the lipid environment contribute to the regulation of organelle fusion (Table 1). Hetero-oligomeric complexes of Mfn1 and 2 Mitofusins are related distantly to dynamin, the large GTPase required for vesicle endocytosis, and, like dynamin, these proteins oligomerize and hydrolyze GTP to
0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.08.006
Review
564
TRENDS in Cell Biology Vol.17 No.11
Figure 1. Fusion and division regulate mitochondrial shape antagonistically. Morphology of yeast mitochondria of wild-type cells and mutants defective for fusion, division or both are shown. (a). Wild-type cells contain tubular mitochondria with occasional branches. (b). Fusion mutants contain fragmented mitochondria owing to ongoing division, whereas (c) division mutants form a single mitochondrial consisting of interconnected tubules. (d). In cells without fusion and division activities, tubular mitochondria, similar to those seen in the wild type, are restored. Images were modified from Sesaki and Jensen, with permission [84].
catalyze the membrane rearrangements that lead to mitochondrial fusion. In addition, recent in vitro membranefusion studies suggest that the yeast mitofusin, Fzo1p, forms complexes that act in trans to fuse mitochondria [24]. Our understanding of mammalian mitochondrial fusion is complicated by the existence of two ubiquitously expressed vertebrate-specific mitofusin isoforms, Mfn1 and Mfn2, which can assemble into homo- and heterooligomers [27]. Although some experiments suggest that these proteins are redundant functionally [27], other experiments indicate that Mfn1 and Mfn2 have specialized functions. For example, Mfn1 exhibits higher GTPase activity and tighter mitochondrial tethering ability than Mfn2 [23]. In addition, Mfn1, but not Mfn2, is required for OPA1 function [28], whereas mutations in Mfn2 cause CMT2A [29].
Detailed analysis of the mutations that cause CMT2A has provided insight into the role of Mfn1–Mfn2 interactions. CMT2A is a progressive neurodegenerative disease characterized by the deterioration of long sensory and motor neuron axons. Characterization of multiple CMT2Aassociated Mfn2 alleles, which are concentrated near and in the GTPase domain, but are also found in the C-terminal oligomerization domain [30], illustrates that many mutant forms of Mfn2 can be rescued although interaction with Mfn1, but not Mfn2. Emphasizing the vital role that hetero-oligomeric complexes of Mfn1 and Mfn2 have during mitochondrial fusion, this functional complementation occurs in trans between Mfn1 and Mfn2 on opposite mitochondria. Furthermore, the specific peripheral neuropathies characteristic of CMT2A might be explained, at least in part, by the ratio of Mfn1 and Mfn2 in different cell types. For instance, cells with high levels of Mfn1, such as cardiomyocytes [31], might be protected from the diseasecausing Mfn2 protein because Mfn1 can form a functional complex with the mutant molecule [30]. However, cells that have lower levels of Mfn1 or that require higher levels of mitochondrial fusion, such as the long peripheral neurons most affected in CMT2A, might be particularly vulnerable to point mutations in Mfn2. With reduced mitochondrial fusion (and therefore a reduction in the distribution of mitochondria), these nerve cells might disperse ATP at the pre- and post-synaptic terminals less efficiently. Studies of a variety of neurodegenerative diseases indicate that appropriately controlled mitochondrial dynamics are crucial for the long-term maintenance of the nervous system [32]. Degradation of Fzo1p (Mfn) Modulating the amount of Mfn protein regulates the extent of mitochondrial fusion. For example, in wild-type yeast
Table 1. Regulation of mitochondrial dynamics Function Fusion
Organism Mammals
Protein Mitofusin 2
OPA1
Yeast
Fzo1p Mgm1p
Division
Mammals
Yeast
www.sciencedirect.com
Drp1
Fis1 Dnm1p
Regulator Bax and Bak Bcl-xL Mitofusin 1 MARCH-V/MITOL MIB Stomatin-like protein 2 PARL Paraplegin Membrane potential Mdm30p Proteasome Pcp1p PAM complex ATP Ups1p cyclin B-CDK cAMP-dependent kinase Sumo-1 SENP5 Bax and Bak Fis1 MARCH-V/MITOL MARCH-V/MITOL Fis1p Mdv1p Caf4p Num1p
Activity Assembly of Mfn2-containing complexes Unknown Complementation of mutant forms of Mfn2 in CMT 2A Possible ubiquitination and degradation Unknown Unkonwn Proteolytic processing Proteolytic processing Proteolytic processing Degradation during growth Degradation during mating Proteolytic processing Proteolytic processing Proteolytic processing Proteolytic processing Phosphorylation during cell cycle Inhibition of assembly and GTPase activitiy by phosphorylation Sumoylaiton Removal of SUMO Mitochondrial association of Drp1 Mitochondrial association of Drp1 Ubiquitination and degradation Ubiquitination and degradation Mitochondrial association of Dnm1p Mitochondrial association of Dnm1p Mitochondrial association of Dnm1p Intracellular dynamics of Dnm1p
Review
TRENDS in Cell Biology
cells, the yeast Mfn homologue, Fzo1p, is turned-over and degraded rapidly by at least two distinct mechanisms [26,33]. During vegetative growth, Fzo1p stability is influenced by the OM F-box-containing protein, Mdm30p. F-boxes are present in subunits of a subset of E3 ubiquitin ligases and are often required for the ubiquitin-conjugating activity of these proteins. Cells expressing a F-box-mutant version of Mdm30p accumulate Fzo1p, contain fragmented mitochondria and exhibit reduced mitochondrial fusion [26], whereas a genome-wide analysis showed that Fzo1p is ubiquitinated [34]. Surprisingly, constitutive Fzo1p turn-over does not require known ubiquitin or proteasome machinery, suggesting that Mdm30p-mediated degradation can occur independently of the proteasome [35,36]. Perhaps yeast cells use autophagy or mitochondrial proteases, such as the m-AAA proteases, to degrade Fzo1p. In contrast to vegetatively growing cells, yeast cells treated with mating pheromone for extended periods of time contain fragmented mitochondria and Fzo1p is degraded by the ubiquitin and proteasome pathway. Interestingly, this process does not appear to involve Mdm30p and it can be blocked when proteasome activity is inhibited pharmacologically [33]. Perhaps proteasome-mediated degradation of yeast Fzo1p favors organelle fragmentation during a-factor-induced cell death, which appears similar to the fragmentation of mitochondria seen in mammalian apoptosis [6,7]. To date, no studies have examined the half-life of Mfns or determined if Mfns are ubiquitinylated. Therefore, it will be interesting to test whether the same rules that govern Fzo1p turn-over impact the function of Mfns in healthy and apoptotic mammalian cells. Assembly of mitofusin (Mfn) complexes by Bcl-2 proteins Mitochondrial fusion and division are regulated during apoptosis. On apoptotic stimulation, the balance between fusion and division is disrupted and the mitochondria fragment [6,7]. Two pro-apoptotic Bcl-2 family members, Bax and Bak, have been implicated in mitochondrial dynamics during programmed cell death and Bax colocalizes with both fusion and fission proteins [37]. Interestingly, Bax and Bak also affect mitochondrial fusion in nonapoptotic cells by regulating the assembly of Mfn2 into high molecular-weight complexes [38]. In the absence of Bak and Bax, Mfn2 complexes become smaller and Mfn2 localization changes from discrete puncta on the organelle surface to a uniform distribution along the mitochondria. Similar, but not identical to Mfn2 deficient cells, Bax/Bak double knockout cells fail to fuse their mitochondria efficiently; as a result, they contain many short mitochondrial tubules. In addition to a role for pro-apoptotic proteins in mitochondrial fusion, two anti-apoptotic Bcl-2 family members – Bcl-xL and Caenorhabditis elegans CED-9 – also promote mitochondrial fusion in mammalian cells by interacting physically with Mfn2 [39]. These findings reveal unexpected non-apoptotic functions for both pro- and antideath Bcl-2 family members and hint that these proteins might exert their reciprocal control over programmed cell death by regulating the assembly of mitochondrial fusion proteins. www.sciencedirect.com
Vol.17 No.11
565
Possible regulation by novel Mfn-binding proteins Recent biochemical studies using mammalian cells have identified two additional Mfn partners, mitofusin-binding protein (MIB) and Stoml2. MIB was isolated as a cytosolic Mfn1-binding protein that associates with the mitochondrial OM and is similar to vesticle amine transferase 1 (VAT-1), an ATPase found in synaptic vesicles predominantly [40]. MIB is a member of the medium-chain dehydrogenase and reductase protein superfamily and it contains a conserved coenzyme-binding domain that might bind and hydrolyze nucleotides. Overexpression of MIB induces mitochondrial fragmentation whereas its knockdown results in a large extension of mitochondrial networks [40]. These phenotypes are consistent with MIB acting either as an activator of mitochondrial division or as an inhibitor of mitochondrial fusion. Consistent with this second possibility, MIB binds the fusion proteins Mfn1 and Mfn2 but not the division protein Drp1. Perhaps MIB uses its nucleotide-binding region to recognize Mfn1–GTP to prevent unproductive nucleotide hydrolysis by Mfn1 until it is fusion complex is assembled or positioned at fusion sites appropriately [40]. Stoml2 is a stomatin-like protein localized to the mitochondrial IMS where it binds Mfn2 [41]. Although knockdown of Stoml2 reduces mitochondrial-membrane potential, this treatment does not alter mitochondrial morphology dramatically. Currently, the role of Stoml2 in mitochondrial dynamics is unknown; however, its prohibitin-like domain might provide some clues to the function of Stoml2. In yeast mitochondria, prohibitins form large oligomeric complexes that perform a variety of functions, including chaperoning mitochondrial (mt)DNA-encoded proteins so that they assemble and insert into the membrane properly. Therefore, Stoml2 might chaperone Mfn2 to help assemble fusion-competent protein complexes. Proteolytic processing of Mgm1p (OPA1) Like the OM Mfn proteins, the machinery required for IM fusion is subjected to modifications that control mitochondrial fusion. Yeast Mgm1p and its mammalian counterpart OPA1 are required for mitochondrial fusion and IM cristae morphology [2,15–17,25]. Mgm1p is processed proteolytically into long (l-Mgm1p) and short forms (s-Mgm1p) inside mitochondria [42–44]. l-Mgm1p is integrated into the IM through its N-terminal transmembrane segment and its mitochondria-targeting presequence is cleaved by the matrix-processing protease. By contrast, s-Mgm1p is processed proteolytically in the IM and then released into the IMS. These two forms are present in approximately equal amounts and seem to perform overlapping as well as distinct roles in maintaining mtDNA, remodeling cristae and fusing mitochondria. Recently, experiments in yeast elucidated the mechanisms that produce l-Mgm1p and s-Mgm1p. s-Mgm1 is generated when Mgm1p is cleaved by the rhomboid-related protease Pcp1p in the IM. The levels of Pcp1p, in addition to the concentration of matrix ATP, regulate the production of s-Mgm1p [43,45]. Cells might use this mechanism to monitor the level of ATP and use this functional readout to inhibit the fusion of defective mitochondria. Ups1p, a protein of evolutionary and lymphoid interest (PRELI), and an evolutionarily conserved
566
Review
TRENDS in Cell Biology Vol.17 No.11
IMS protein, regulates the relative amounts of long and short forms of Mgm1p by controlling the insertion of Mgm1p’s Pcp1p-sensitive rhomboid-cleavage site into the IM [46]. Similar events control the processing of OPA1 in mammals. Similar to Mgm1p processing, OPA1 cleavage reflects the energy level in mitochondria and can be regulated by the membrane potential across the IM [47]. However, the identity of the protease that cleaves OPA1 is controversial. The mammalian homologue of yeast Pcp1p, called presenilin-associated rhomboid-like protein (PARL), has been implicated in OPA1 proteolysis. PARL does not appear to regulate the function of OPA1 in mitochondrial fusion, although it does influence the ability of Opa1p to remodel cristae [48,49]. Supporting its role in mitochondrial morphology, phosphorylation of PARL affects mitochondrial shape; however, it remains to be determined whether the phosphorylation of PARL modulates Opa1 cleavage [50]. At least three other proteins have been associated with OPA1 processing. The two m-AAA proteases AFG3L2 [51] and Paraplegin [52] and the i-AAA protease Yme1 [53,54] process OPA1 and it is likely that these three AAA proteases might combine in various ratios to insure OPA1 cleavage under a variety of cellular conditions [51]. Furthermore, because mammalian cells contain at least eight different splice variants of OPA1 [55], it is likely that many versions of OPA1 contribute to mitochondrial remodeling. Clearly, further investigations into the proteins and mechanisms that contribute to the functions of OPA1 are warranted. Lipid modification by phospholipase D The lipid composition of the mitochondrial membrane bilayers can influence membrane remodeling. Indeed, some lipids, such as phosphatidic acid (PA), are particularly fusogenic and seem to contribute to stereotypical changes in membrane curvature that thermodynamically favor deformation and bilayer mixing [56]. Choi et al. [57] identified a mitochondria-targeted member of the phospholipase D (PLD) protein family that is required for mitochondrial fusion. This highly divergent lipase, named MitoPLD, catalyzes the synthesis of PA from the mitochondria-enriched lipid, cardiolipin. The catalytic site of MitoPLD is located on the cytosolic surface of the OM. Because the substrate for this protein, cardiolipin, is located mainly in the IM, MitoPLD might catalyze fusion at contact sites, places where the IM and OM are closely apposed, which are thought to provide conduits for lipid and protein transfer from one membrane to the other [17,58–60]. Many questions about how MitoPLD functions remain. For instance, does its hydrolysis of cardiolipin activate the membrane-anchored fusion proteins or does it simply alter membrane curvature to favor lipid mixing? The isolation and characterization of this interesting protein has provided the first step towards understanding how the local lipid environment can facilitate mitochondrial dynamics. Regulation of mitochondrial division Similar to mitochondrial fusion, mitochondrial division requires a dynamin-related GTPase, called Dnm1p in www.sciencedirect.com
yeast and Drp1 in mammals [61–65]. In contrast to the integral membrane-fusion proteins, these proteins shuttle between the cytosol and the surface of mitochondria. Dnm1p (Drp1) is recruited to the mitochondrial surface and assembles into oligomeric complexes, which are thought to wrap around mitochondria surface by a variety of receptors – Mdv1, Caf4, and Fis – and then assembles into oligomeric complexes [66–69]. As these complexes assemble, it has been proposed that they wrap around the mitochondrial tubules like spirals that constrict and eventually divide mitochondria [70]. Below we summarize recent findings that have uncovered new regulatory mechanisms that control mitochondrial fission in response to a variety of cellular events, including cell division, metabolic flux and cell differentiation. Most of these processes seem to regulate the localization, dynamics and activity of Dnm1p (Drp1) (Table 1). Phosphorylation of Drp1 Mitochondrial division is coordinated with the cell cycle in higher eukaryotes. Recent experiments using cultured human cells showed elegantly that mitochondrial scission is induced at the onset of mitosis, leading to partial fragmentation of mitochondria [71]. Revealing a direct link between the cell-cycle and the mitochondrial-division machinery, this burst of mitochondrial division is correlated with the cyclinB–cyclin-dependant kinase (CDK1dependent) phosphorylation of Drp1. This is the first demonstration that the addition of a phosphate moiety to Drp1 regulates its activity and in vitro assays using purified proteins coupled with cell-culture experiments indicate that the most potent mitotic phosphorylation event occurs on a serine residue in the carboxyl-terminal GTPase-effecter domain (GED) of Drp1. Another recent study showed that cyclic-AMP-kinase-dependent phosphorylation of a different serine in the GED can decrease the GTPase activity of Drp1 by inhibiting the intramolecular interactions known to increase the GTPase activity of Drp1 [72]. Although these initial studies indicate that Drp1 phosphorylation can modulate the frequency of mitochondrial division, it remains to be determined if fissioncompetent Drp1 is always phosphorylated or if this is a mechanism exploited only during the cell cycle. In addition, it will be useful to learn if the phosphorylation state of Dnm1p influences mitochondrial division in yeast, especially during mitosis. Ubiquitinylation of Drp1 Ubiquitinylation regulates mitochondrial division. Experiments using yeast mitochondria have raised the possibility that ubiquitin conjugation to mitochondria controls a variety of organelle processes, including mitochondrial dynamics and inheritance [73]. The most convincing evidence to date that ubiquitinylation controls mitochondrial division comes from three independent investigations of the membrane-associated RING-CH (MARCH-V (MITOL)) E3 ubiquitin ligase in mammalian cells. This OM-anchored protein is capable of binding and modifying Drp1 and its OM anchor, Fis1 [74–76]. Although the data from these studies support conflicting functions for MARCH-V – either as an activator or an inhibitor of fission – it is clear
Review
TRENDS in Cell Biology
that binding between Drp1 and MARCH-V influences mitochondrial dynamics. The most recent work [76] suggests that MARCH-V ubiquitinylates Drp1 but does not control the stability of Drp1. Instead, Drp1 ubiquitinylation seems to regulate the kinetics of Drp1 binding to the mitochondrial surface. In this instance, ubiquitin conjugation might regulate the subcellular trafficking, assembly of Drp1 and influence the rate of mitochondrial division [76]. Much remains to be learned about the stability and trafficking of Drp1. In particular, it will be interesting to ascertain if particular signals, such as cellular stresses or cell-cycle progression, influence the frequency and amount of ubiquitinylated Drp1 and if the activity of the yeast cousin of Drp1, Dnm1p, is modified by ubiquitin. Sumoylation of Drp1 The small ubiquitin-like modifier (SUMO) is highly conserved from yeast to humans and can be conjugated to a wide variety of proteins in a manner similar to ubiquitin. Unlike ubiquitinylation, which often leads to degradation of substrates, sumoylation usually alters the subcellular localization of target proteins or protects them from ubiquitin-mediated destruction. Drp1 pull-down experiments identified SUMO-1 and its conjugating enzyme Ubc9 as Drp1-binding partners [77]. Confirming that these two proteins form a functional complex that binds and modifies Drp1, sumoylated Drp1 has been isolated from mammalian cells [77]. Consistent with the idea that sumoylated Drp1 promotes mitochondrial division, overexpressing SUMO1 or silencing sentrin/SUMO-specific protease (SENP5), the protease that removes SUMO from Drp1, yields cells with many small mitochondria [77,78]. Interestingly, SENP5 is also a key player in cell-cycle progression [79] and, similar to the cyclinB–CDK1 dependent phosphorylation of Drp1, the interactions among Drp1, SUMO and SENP5 might help
Vol.17 No.11
567
to coordinate mitochondrial division and the cell cycle to insure even segregation of mitochondria. Regulation of Drp1 association with mitochondria by Bax and Bak Sumoylation of Drp1 also appears to influence the integration of pro-apoptotic signals and mitochondrial division. Recent experiments illustrate that normal cycling of Drp1 on and off the organelle surface is arrested during apoptosis, such that Drp1 accumulates on the mitochondria [80]. In this scenario, Drp1 is recruited to the mitochondrial membrane independent of its well characterized membrane anchor, Fisp1. In addition, Drp1 becomes sumoylated in a Bax- and Bak-dependent manner, suggesting that these pro-apoptotic proteins enhance the mitochondrial association of Drp1 through sumoylation. These events immediately precede apoptosis, which raises the possibility that sumoylation might alter the activity of Drp1 as a result of apoptotic signals thereby contributing to mitochondrial fragmentation during programmed cell death. Regulation of Dnm1p dynamics by Num1p In addition to the mechanisms that modulate mitochondrial division in mammalian cells, many early studies exploited the genetic potential of yeast to understand how mitochondria divide [3,81]. The latest of these studies, which searched for high-copy suppressors of mutant Dnm1p (the yeast orthologue of Drp1), found that the cortically localized multidomain Num1 protein affects mitochondrial division. Many previous studies demonstrated that Num1p controls nuclear migration and inheritance through its association with components of both microtubule and actin cytoskeletons [82]. In addition to this well established role for Num1p, it also participates in the division and inheritance of mitochondria. Reminiscent of the
Figure 2. Regulation of mitochondrial fusion and division. Schematic of organelle dynamics highlights many of the processes that regulate mitochondrial dynamics and illustrates the consequences of unbalanced fusion or division. Defects in mitochondrial fusion (unbalanced division) lead to a variety of pathologies, including CharcotMarie-Tooth Disease type 2A (CMT type 2A) and dominant optic atrophy (DOA). In addition, excessive mitochondrial fragmentation accompanies apoptosis. Defects in mitochondrial division (unbalanced fusion) cause defects in neural development and Charcot-Marie-Tooth Disease type 4 (CMT type 4). www.sciencedirect.com
568
Review
TRENDS in Cell Biology Vol.17 No.11
control that MARCH-V and SUMO-1 exert over Drp1 trafficking, Num1p appears to regulate mitochondrial division by influencing the dynamic interactions of Dnm1p with mitochondria. Not only is Num1p important for mitochondrial division but it also cooperates with Dnm1p to insure mitochondrial inheritance during mitosis [83]. Given the intimate association of Num1p with the cytoskeleton, its function in nuclear migration and its physical interaction with Dnm1p, Num1p is positioned perfectly to coordinate organelle division and inheritance. Whether similar proteins capable of linking organelle division with inheritance exist in mammalian cells awaits further inquiry. Conclusions and perspectives The dynamic equilibrium between fusion and division enables rapid and robust changes in mitochondrial structure and function by stimulating one activity and/or repressing the other (Figure 2). Reinforcing the importance of the antagonistic relationship between mitochondrial fusion and division, some of the factors that influence mitochondrial fusion, such as Bcl-2 proteins and ubiquitin, also have the ability to modulate the opposing process of division. Moreover, identification and study of the mechanisms that integrate cellular processes, such as cell division and apoptosis, with mitochondrial remodeling, have yielded glimpses into how a cell’s physiological context can influence mitochondrial morphology and function. Whether specific cell types have evolved unique mechanisms to adapt their mitochondrial morphology in response to intrinsic and extrinsic signals remains an open question. Recent publications have reported on many of the signals that regulate mitochondrial dynamics, including the diverse processes of phosphorylation, ubiquitinylation, proteolytic processing, protein trafficking, lipid remodeling and assembly of functional protein complexes, however, we know very little about how all of these signals are connected. Continued investigation into the proteins and lipids that regulate mitochondrial dynamics will contribute new insight to our understanding of how mitochondrial fusion and division are coordinated with the developmental and metabolic needs of eukaryotic organisms. Acknowledgements This work was supported by Damon Runyon Cancer Research Foundation (DRG1877–05) (K.L.C), Uehara Memorial Foundation (Y.T.), National Institutes of Health (R.E.J.), Johns Hopkins University and American Heart Association (H.S.).
References 1 Osteryoung, K.W. and Nunnari, J. (2003) The division of endosymbiotic organelles. Science 302, 1698–1704 2 Griparic, L. et al. (2004) Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792–18798 3 Okamoto, K. and Shaw, J.M. (2005) Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu. Rev. Genet. 39, 503–536 4 Chung, S. et al. (2007) Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat. Clin. Pract. Cardiovasc. Med. 4 (Suppl. 1), S60–S67 5 Li, Z. et al. (2004) The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873–887 6 Cereghetti, G.M. and Scorrano, L. (2006) The many shapes of mitochondrial death. Oncogene 25, 4717–4724 www.sciencedirect.com
7 Youle, R.J. and Karbowski, M. (2005) Mitochondrial fission in apoptosis. Nat. Rev. Mol. Cell Biol. 6, 657–663 8 Olichon, A. et al. (2006) Mitochondrial dynamics and disease, OPA1. Biochim. Biophys. Acta 1763, 500–509 9 Chan, D.C. (2006) Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241–1252 10 Waterham, H.R. et al. (2007) A lethal defect of mitochondrial and peroxisomal fission. N. Engl. J. Med. 356, 1736–1741 11 Cuesta, A. et al. (2002) The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal CharcotMarie-Tooth type 4A disease. Nat. Genet. 30, 22–25 12 Niemann, A. et al. (2005) Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease. J. Cell Biol. 160, 1067–1078 13 Chan, D.C. (2006) Mitochondrial fusion and fission in mammals. Annu. Rev. Cell Dev. Biol. 22, 79–99 14 Meeusen, S.L. and Nunnari, J. (2005) How mitochondria fuse. Curr. Opin. Cell Biol. 17, 389–394 15 Olichon, A. et al. (2003) Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. J. Biol. Chem. 278, 7743–7746 16 Wong, E.D. et al. (2000) The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria. J. Cell Biol. 151, 341–352 17 Sesaki, H. et al. (2003) Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane. Mol. Biol. Cell 14, 2342–2356 18 Santel, A. and Fuller, M.T. (2001) Control of mitochondrial morphology by a human mitofusin. J. Cell Sci. 114, 867–874 19 Hermann, G.J. et al. (1998) Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p. J. Cell Biol. 143, 359–373 20 Rapaport, D. et al. (1998) Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae. J. Biol. Chem. 273, 20150–20155 21 Hales, K.G. and Fuller, M.T. (1997) Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90, 121–129 22 Koshiba, T. et al. (2004) Structural basis of mitochondrial tethering by mitofusin complexes. Science 305, 858–862 23 Ishihara, N. et al. (2004) Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity. J. Cell Sci. 117, 6535–6546 24 Meeusen, S. et al. (2004) Mitochondrial fusion intermediates revealed in vitro. Science 305, 1747–1752 25 Meeusen, S. et al. (2006) Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Cell 127, 383–395 26 Fritz, S. et al. (2003) Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast. Mol. Biol. Cell 14, 2303–2313 27 Chen, H. et al. (2003) Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J. Cell Biol. 160, 189–200 28 Cipolat, S. et al. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U. S. A. 101, 15927–15932 29 Zuchner, S. et al. (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat. Genet. 36, 449–451 30 Detmer, S.A. and Chan, D.C. (2007) Complementation between mouse Mfn1 and Mfn2 protects mitochondrial fusion defects caused by CMT2A disease mutations. J. Cell Biol. 176, 405–414 31 Santel, A. et al. (2003) Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells. J. Cell Sci. 116, 2763–2774 32 Mandemakers, W. et al. (2007) A cell biological perspective on mitochondrial dysfunction in Parkinson disease and other neurodegenerative diseases. J. Cell Sci. 120, 1707–1716 33 Neutzner, A. and Youle, R.J. (2005) Instability of the mitofusin Fzo1 regulates mitochondrial morphology during the mating response of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 280, 18598–18603 34 Hitchcock, A.L. et al. (2003) A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum
Review
35
36
37
38 39 40
41
42
43 44
45
46
47
48 49
50
51
52 53
54 55 56 57 58
TRENDS in Cell Biology
degradation machinery. Proc. Natl. Acad. Sci. U. S. A. 100, 12735– 12740 Durr, M. et al. (2006) Nonredundant roles of mitochondria-associated F-box proteins Mfb1 and Mdm30 in maintenance of mitochondrial morphology in yeast. Mol. Biol. Cell 17, 3745–3755 Escobar-Henriques, M. et al. (2006) Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1. J. Cell Biol. 173, 645–650 Karbowski, M. et al. (2002) Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J. Cell Biol. 159, 931–938 Karbowski, M. et al. (2006) Role of Bax and Bak in mitochondrial morphogenesis. Nature 443, 658–662 Delivani, P. et al. (2006) Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics. Mol. Cell 21, 761–773 Eura, Y. et al. (2006) Identification of a novel protein that regulates mitochondrial fusion by modulating mitofusin (Mfn) protein function. J. Cell Sci. 119, 4913–4925 Hajek, P. et al. (2007) Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2. J. Biol. Chem. 282, 5670–5681 Sesaki, H. et al. (2003) Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependentmanner, but remain competent for mitochondrial fusion. Biochem. Biophys. Res. Commun. 308, 276–283 McQuibban, G.A. et al. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541 Herlan, M. et al. (2003) Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J. Biol. Chem. 278, 27781–27788 Herlan, M. et al. (2004) Alternative topogenesis of Mgm1 and mitochondrial morphology depend on ATP and a functional import motor. J. Cell Biol. 165, 167–173 Sesaki, H. et al. (2006) Ups1p, a conserved intermembrane space protein, regulates mitochondrial shape and alternative topogenesis of Mgm1p. J. Cell Biol. 173, 651–658 Duvezin-Caubet, S. et al. (2006) Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology. J. Biol. Chem. 281, 37972–37979 Frezza, C. et al. (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126, 177–189 Cipolat, S. et al. (2006) Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126, 163–175 Jeyaraju, D.V. et al. (2006) Phosphorylation and cleavage of presenilinassociated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology. Proc. Natl. Acad. Sci. U. S. A. 103, 18562–18567 Duvezin-Caubet, S. et al. (2007) OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria. Mol. Biol. Cell 18, 3582–3590 Ishihara, N. et al. (2006) Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. EMBO J. 25, 2966–2977 Song, Z. et al. (2007) OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L. J. Cell Biol. 178, 749–755 Griparic, L. et al. (2007) Regulation of the mitochondrial dynaminrelated protein Opa1 by proteolytic cleavage. J. Cell Biol. 178, 757–764 Delettre, C. et al. (2002) OPA1 (Kjer type) dominant optic atrophy: a novel mitochondrial disease. Mol. Genet. Metab. 75, 97–107 Chernomordik, L.V. et al. (2006) Membranes of the world unite! J. Cell Biol. 175, 201–207 Choi, S.Y. et al. (2006) A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis. Nat. Cell Biol. 8, 1255–1262 Fritz, S. et al. (2001) Connection of the mitochondrial outer and inner membranes byFzo1 is critical for organellar fusion. J. Cell Biol. 152, 683–692
www.sciencedirect.com
Vol.17 No.11
569
59 Sesaki, H. and Jensen, R.E. (2004) Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion. J. Biol. Chem. 279, 28298–28303 60 Wong, E.D. et al. (2003) The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion. J. Cell Biol. 160, 303–311 61 Labrousse, A.M. et al. (1999) C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane. Mol. Cell 4, 815–826 62 Smirnova, E. et al. (2001) Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol. Biol. Cell 12, 2245– 2256 63 Pitts, K.R. et al. (2004) Mitochondria-specific function of the dynamin family protein DLP1 is mediated by its C-terminal domains. J. Biol. Chem. 279, 50286–50294 64 Sesaki, H. and Jensen, R.E. (1999) Division versus fusion: Dnm1p and Fzo1 pantagonistically regulate mitochondrial shape. J. Cell Biol. 147, 699–706 65 Bleazard, W. et al. (1999) The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1, 298–304 66 Griffin, E.E. et al. (2005) The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria. J. Cell Biol. 170, 237–248 67 Cerveny, K.L. et al. (2001) Division of mitochondria requires a novel DNM1interacting protein, Net2p. Mol. Biol. Cell 12, 309–312 68 Tieu, Q. and Nunnari, J. (2000) Mdv1p Is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division. J. Cell Biol. 151, 353–366 69 Mozdy, A.D. et al. (2000) Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p. J. Cell Biol. 151, 367–380 70 Ingerman, E. et al. (2005) Dnm1 forms spirals that are structurally tailored to fit mitochondria. J. Cell Biol. 170, 1021–1027 71 Taguchi, N. et al. (2007) Mitotic phosphorylation of dynamin-related GTPase Drp1participates in mitochondrial fission. J. Biol. Chem. 282, 11521–11529 72 Chang, C.R. and Blackstone, C. (2007) Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology. J. Biol. Chem. 282, 21583–21587 73 Escobar-Henriques, M. and Langer, T. (2006) Mitochondrial shaping cuts. Biochim. Biophys. Acta 1763, 422–429 74 Nakamura, N. et al. (2006) MARCH-V is a novel mitofusin 2- and Drp1binding protein able to change mitochondrial morphology. EMBO Rep. 7, 1019–1022 75 Yonashiro, R. et al. (2006) A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J. 25, 3618–3626 76 Karbowski, M. et al. (2007) The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division. J. Cell Biol. 178, 71–84 77 Harder, Z. et al. (2004) Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission. Curr. Biol. 14, 340–345 78 Zunino, R. et al. (2007) The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. J. Cell Sci. 120, 1178–1188 79 Di Bacco, A. and Gill, G. (2006) SUMO-specific proteases and the cell cycle. An essential role for SENP5 in cell proliferation. Cell Cycle 5, 2310–2313 80 Wasiak, S. et al. (2007) Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death. J. Cell Biol. 177, 439–450 81 Jensen, R.E. et al. (2000) Yeast mitochondrial dynamics: fusion, division, segregation, and shape. Microsc. Res. Tech. 51, 573–583 82 Bloom, K. (2001) Nuclear migration: cortical anchors for cytoplasmic dynein. Curr. Biol. 11, R326–R329 83 Cerveny, K.L. et al. (2007) Yeast mitochondrial division and distribution require the cortical num1 protein. Dev. Cell 12, 363–375 84 Sesaki, H. and Jensen, R.E. (1999) Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J. Cell Biol. 147, 699–706