The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria

The mitochondrial division machinery regulates mitochondrial dynamics and consists of Fis1p, Mdv1p, and Dnm1p. Mitochondrial division relies on the recruitment of the dynamin-related protein Dnm1p to mitochondria. Dnm1p recruitment depends on the mitochondrial outer membrane protein Fis1p. Mdv1p interacts with Fis1p and Dnm1p, but is thought to act at a late step during fission because Mdv1p is dispensable for Dnm1p localization. We identify the WD40 repeat protein Caf4p as a Fis1p-associated protein that localizes to mitochondria in a Fis1p-dependent manner. Caf4p interacts with each component of the fission apparatus: with Fis1p and Mdv1p through its NH2-terminal half and with Dnm1p through its COOH-terminal WD40 domain. We demonstrate that mdv1delta yeast contain residual mitochondrial fission due to the redundant activity of Caf4p. Moreover, recruitment of Dnm1p to mitochondria is disrupted in mdv1delta caf4delta yeast, demonstrating that Mdv1p and Caf4p are molecular adaptors that recruit Dnm1p to mitochondrial fission sites. Our studies support a revised model for assembly of the mitochondrial fission apparatus.     

The WD repeat protein,Mdv1p,functions as a molecular adaptor by interacting with Dnm1p and Fis1p during mitochondrial fission

Yeast mitochondrial fission is a multistep process during which the dynamin-related GTPase, Dnm1p, assembles into punctate structures that associate with the outer mitochondrial membrane and mediate mitochondrial division. Steps in the Dnm1p-dependent process of fission are regulated by the actions of the WD repeat protein, Mdv1p, and the mitochondrial outer membrane protein, Fis1p. Our previous studies suggested a model where Mdv1p functions to regulate fission at a post-Dnm1p assembly step and Fis1p functions at two distinct steps, at an early point, to regulate Dnm1p assembly, and later, together with Mdv1p, to facilitate Dnm1p-dependent mitochondrial fission. To test this model, we have examined the physical and functional relationship between Mdv1p and Fis1p and present genetic, biochemical, and two-hybrid data indicating that a Fis1p-Mdv1p complex is required to regulate mitochondrial fission. To further define the role of Mdv1p in fission, we examined the structural features of Mdv1p required for its interactions with Dnm1p and Fis1p. Data from two-hybrid analyses and GFP-tagged domains of Mdv1p indicate that it contains two functionally distinct domains that enable it to function as a molecular adaptor to regulate sequential interactions between Dnm1p and Fis1p and catalyze a rate-limiting step in mitochondrial fission.     

Fis1 deficiency selects for compensatory mutations responsible for cell death and growth control defects

Genetic mutations affecting mitochondrial fission and fusion proteins cause human neurological disorders, but are assumed to be well tolerated in yeast. The conserved mitochondrial fission protein Dnm1/Drp1 is required for normal mitochondrial division, but also promotes cell death in mammals and yeast. Fis1, an outer mitochondrial membrane-anchored receptor for Dnm1/Drp1, also can promote cell death in mammals, but appears to have prosurvival activity in yeast. Here we report that deletion of the FIS1 gene in yeast consistently results in acquisition of a secondary mutation that confers sensitivity to cell death. In several independently derived FIS1 knockouts, tiling arrays and genomic sequencing identified the secondary mutation as a premature termination in the same stress-response gene, WHI2. The WHI2 mutation rescues the mitochondrial respiratory defect (petite formation) caused by FIS1 deficiency, but also causes a failure to suppress cell growth during amino-acid deprivation. Thus, loss of Fis1 drives the selection for specific compensatory mutations that confer defective growth control and cell death regulation, characteristic of human tumor cells. The important long-term survival function of Fis1 that is compensated by WHI2 mutation appears to be independent of fission factor Dnm1/Drp1 and its adaptor Mdv1, but may be mediated through a second adaptor Caf4, as WHI2 is also mutated in a CAF4 knockout.     

Dimeric Dnm1-G385D interacts with Mdv1 on mitochondria and can be stimulated to assemble into fission complexes containing Mdv1 and Fis1

Interactions between yeast Dnm1p, Mdv1p, and Fis1p are required to form fission complexes that catalyze division of the mitochondrial compartment. During the formation of mitochondrial fission complexes, the Dnm1p GTPase self-assembles into large multimeric complexes on the outer mitochondrial membrane that are visualized as punctate structures by fluorescent labeling. Although it is clear that Fis1p.Mdv1p complexes on mitochondria are required for the initial recruitment of Dnm1p, it is not clear whether Dnm1p puncta assemble before or after this recruitment step. Here we show that the minimum oligomeric form of cytoplasmic Dnm1p is a dimer. The middle domain mutant protein Dnm1G385Dp forms dimers in vivo but fails to assemble into punctate structures. However, this dimeric mutant stably interacts with Mdv1p on the outer mitochondrial membrane, demonstrating that assembly of stable Dnm1p multimers is not required for Dnm1p-Mdv1p association or for mitochondrial recruitment of Dnm1p. Dnm1G385Dp is reported to be a terminal dimer in vitro. We describe conditions that allow assembly of Dnm1G385Dp into functional fission complexes on mitochondria in vivo. Using these conditions, we demonstrate that multimerization of Dnm1p is required to promote reorganization of Mdv1p from a uniform mitochondrial localization into punctate fission complexes. Our studies also reveal that Fis1p is present in these assembled fission complexes. Based on our results, we propose that Dnm1p dimers are initially recruited to the membrane via interaction with Mdv1p.Fis1p complexes. These dimers then assemble into multimers that subsequently promote the reorganization of Mdv1p into punctate fission complexes.     

Peroxisome fission in Hansenula polymorpha requires Mdv1 and Fis1, two proteins also involved in mitochondrial fission

We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae , also function in peroxisome proliferation. Deletion of MDV1 , CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome-inducing growth conditions, most likely related to the fact that Vps1 – and not Dnm1 – is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha , which has only a Dnm1-dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha . In wild-type H. polymorpha , cells Dnm1–mCherry and green fluorescent protein (GFP)–Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP–Mdv1 spots – and related to this normal organelle fission – is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP–Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1–GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.     

Crystal structure of mitochondrial fission complex reveals scaffolding function for mitochondrial division 1 (Mdv1) coiled coil

The mitochondrial fission machinery is best understood in the yeast Saccharomyces cerevisiae, where Fis1, Mdv1, and Dnm1 are essential components. Fis1 is a mitochondrial outer membrane protein that recruits the dynamin-related GTPase Dnm1 during the fission process. This recruitment occurs via Mdv1, which binds both Fis1 and Dnm1 and therefore functions as a molecular adaptor linking the two molecules. Mdv1 has a modular structure, consisting of an N-terminal extension that binds Fis1, a central coiled coil for dimerization, and a C-terminal WD40 repeat region that binds Dnm1. We have solved the crystal structure of a dimeric Mdv1-Fis1 complex that contains both the N-terminal extension and coiled-coil regions of Mdv1. Consistent with previous studies, Mdv1 binds Fis1 through a U-shaped helix-loop-helix motif, and dimerization of the Mdv1-Fis1 complex is mediated by the antiparallel coiled coil of Mdv1. However, the complex is surprisingly compact and rigid due to two additional contacts mediated by the surface of the Mdv1 coiled coil. The coiled coil packs against both Fis1 and the second helix of the Mdv1 helix-loop-helix motif. Mutational analyses showed that these contacts are important for mitochondrial fission activity. These results indicate that, in addition to dimerization, the unusually long Mdv1 coiled coil serves a scaffolding function to stabilize the Mdv1-Fis1 complex.     

The role of Fis1p-Mdv1p interactions in mitochondrial fission complex assembly

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)-like fold and a short NH(2)-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p-Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH(2)-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.     

Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p

Yeast Dnm1p is a soluble, dynamin-related GTPase that assembles on the outer mitochondrial membrane at sites where organelle division occurs. Although these Dnm1p-containing complexes are thought to trigger constriction and fission, little is known about their composition and assembly, and molecules required for their membrane recruitment have not been isolated. Using a genetic approach, we identified two new genes in the fission pathway, FIS1 and FIS2. FIS1 encodes a novel, outer mitochondrial membrane protein with its amino terminus exposed to the cytoplasm. Fis1p is the first integral membrane protein shown to participate in a eukaryotic membrane fission event. In a related study (Tieu, Q., and J. Nunnari. 2000. J. Cell Biol. 151:353-365), it was shown that the FIS2 gene product (called Mdv1p) colocalizes with Dnm1p on mitochondria. Genetic and morphological evidence indicate that Fis1p, but not Mdv1p, function is required for the proper assembly and distribution of Dnm1p-containing fission complexes on mitochondrial tubules. We propose that mitochondrial fission in yeast is a multi-step process, and that membrane-bound Fis1p is required for the proper assembly, membrane distribution, and function of Dnm1p-containing complexes during fission.     

Molecular architecture of a dynamin adaptor: implications for assembly of mitochondrial fission complexes

Recruitment and assembly of some dynamin-related guanosine triphosphatases depends on adaptor proteins restricted to distinct cellular membranes. The yeast Mdv1 adaptor localizes to mitochondria by binding to the membrane protein Fis1. Subsequent Mdv1 binding to the mitochondrial dynamin Dnm1 stimulates Dnm1 assembly into spirals, which encircle and divide the mitochondrial compartment. In this study, we report that dimeric Mdv1 is joined at its center by a 92-Å antiparallel coiled coil (CC). Modeling of the Fis1–Mdv1 complex using available crystal structures suggests that the Mdv1 CC lies parallel to the bilayer with N termini at opposite ends bound to Fis1 and C-terminal β-propeller domains (Dnm1-binding sites) extending into the cytoplasm. A CC length of appropriate length and sequence is necessary for optimal Mdv1 interaction with Fis1 and Dnm1 and is important for proper Dnm1 assembly before membrane scission. Our results provide a framework for understanding how adaptors act as scaffolds to orient and stabilize the assembly of dynamins on membranes.     

Mdv1 interacts with assembled dnm1 to promote mitochondrial division

The dynamin-related GTPase, Dnm1, self-assembles into punctate structures that are targeted to the outer mitochondrial membrane where they mediate mitochondrial division. Post-targeting, Dnm1-dependent division is controlled by the actions of the WD repeat protein, Mdv1, and the mitochondrial tetratricopeptide repeat-like outer membrane protein, Fis1. Our previous studies suggest a model where at this step Mdv1 functions as an adaptor linking Fis1 with Dnm1. To gain insight into the exact role of the Fis1.Mdv1.Dnm1 complex in mitochondrial division, we performed a structure-function analysis of the Mdv1 adaptor. Our analysis suggests that dynamic interactions between Mdv1 and Dnm1 play a key role in division by regulating Dnm1 self-assembly.     

线粒体分裂、融合与细胞凋亡

线粒体是高度动态变化的细胞器,其在细胞内不断分裂、融合并形成网状结构。线粒体的分裂和融合是由多种蛋白质精确调控完成的。Drp1／Dnm1p,Fis1／Fis1p,Caf4p和Mdv1p参与线粒体分裂的调控;Mfn1／2／Fzo1p控制线粒体外膜的融合,而Mgm1p／OPA1则参与线粒体内膜的融合。在细胞凋亡过程中线粒体片段化,网状结构被破坏,线粒体嵴发生重构,抑制这一过程可以部分抑制细胞色素c的释放和细胞凋亡。线粒体形态对于细胞维持正常生理代谢和机体发育起着重要的作用,一旦出现障碍会导致严重的疾病。     

A novel motif in the yeast mitochondrial dynamin Dnm1 is essential for adaptor binding and membrane recruitment

To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms.     

Direct binding of the dynamin-like GTPase, Dnm1, to mitochondrial dynamics protein Fis1 is negatively regulated by the Fis1 N-terminal arm

Recruitment of a dynamin-like GTPase (Drp1/Dlp1/Dnm1) to membranes requires the mitochondrial dynamics protein Fis1. Mdv1 has been proposed to act as an adaptor between Fis1 and Dnm1 in Saccharomyces cerevisiae. We show that S. cerevisiae Fis1 binds directly to Dnm1 and to Mdv1. Two Fis1 regions have been previously implicated in Mdv1 recruitment: an N-terminal "arm" and a concave surface formed by evolutionarily conserved residues in the tetratricopeptide repeat domain. Perturbing either Fis1 region does not affect Mdv1 binding, but both regions influence Dnm1 binding. Fis1 lacking its N-terminal arm binds tightly to Dnm1, and binding is abolished by mutations to the Fis1 concave surface. The Fis1-Dnm1 interaction decreases more than 100-fold in the presence of the Fis1 arm, suggesting that the arm acts in an autoinhibitory manner to restrict access to the Dnm1 binding site on Fis1. Our data indicate that the concave surface of the Fis1 tetratricopeptide repeat-like domain is evolutionarily conserved to bind the dynamin-like GTPase Dnm1 and not Mdv1 as previously predicted.     

The solution structure of human mitochondria fission protein Fis1 reveals a novel TPR-like helix bundle

Fis1 in yeast localizes to the outer mitochondrial membrane and facilitates mitochondrial fission by forming protein complexes with Dnm1 and Mdv1. Fis1 orthologs exist in higher eukaryotes, suggesting that they are functionally conserved. In the present study, we cloned the human Fis1 ortholog that was predicted in a database, and determined the protein structure using NMR spectroscopy. Following a flexible N-terminal tail, six alpha-helices connected with short loops construct a single core domain. The C-terminal tail containing a transmembrane segment appears to be disordered. In the core domain, each of two sequentially adjacent helices forms a hairpin-like conformation, resulting in a six helix assembly forming a slightly twisted slab similar to that of a tandem array of tetratrico-peptide repeat (TPR) motif folds. Within this TPR-like core domain, no significant sequence similarity to the typical TPR motif is found. The structural analogy to the TPR-containing proteins suggests that Fis1 binds to other proteins at its concave hydrophobic surface. A simple composition of Fis1 comprised of a binding domain and a transmembrane segment indicates that the protein may function as a molecular adaptor on the mitochondrial outer membrane. In HeLa cells, however, increased levels in mitochondria-associated Fis1 did not result in mitochondrial translocation of Drp1, a potential binding partner of Fis1 implicated in the regulation of mitochondrial fission, suggesting that the interaction between Drp1 and Fis1 is regulated.     

Novel structure of the N terminus in yeast Fis1 correlates with a specialized function in mitochondrial fission

Mitochondrial fission is facilitated by a multiprotein complex assembled at the division site. The required components of the fission machinery in Saccharomyces cerevisiae include Dnm1, Fis1, and Mdv1. In the present study, we determined the protein structure of yeast Fis1 using NMR spectroscopy. Although the six alpha-helices, as well as their folding, in the yeast Fis1 structure are similar to those of the tetratricopeptide repeat (TPR) domains of the human Fis1 structure, the two structures differ in their N termini. The N-terminal tail of human Fis1 is flexible and unstructured, whereas a major segment of the longer N terminus of yeast Fis1 is fixed to the concave face formed by the six alpha-helices in the TPR domains. To investigate the role of the fixed N terminus, exogenous Fis1 was expressed in yeast lacking the endogenous protein. Expression of yeast Fis1 protein rescued mitochondrial fission in delta fis1 yeast only when the N-terminal TPR binding segment was left intact. The presence of this segment is also correlated to the recruitment of Mdv1 to mitochondria. The conformation of the N-terminal segment embedded in the TPR pocket indicates an intra-molecular regulation of Fis1 bioactivity. Although the TPR-like helix bundle of Fis1 mediates the interaction with Dnm1 and Mdv1, the N terminus of Fis1 is a prerequisite to recruit Mdv1 to facilitate mitochondrial fission.     

Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division

Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p, which assembles on the mitochondrial outer membrane into punctate structures associated with sites of membrane constriction and fission. We have identified additional nuclear genes required for mitochondrial fission, termed MDV (for mitochondrial division). MDV1 encodes a predicted soluble protein, containing a coiled-coil motif and seven COOH-terminal WD repeats. Genetic and two-hybrid analyses indicate that Mdv1p interacts with Dnm1p to mediate mitochondrial fission. In addition, Mdv1p colocalizes with Dnm1p in fission-mediating punctate structures on the mitochondrial outer membrane. Whereas localization of Mdv1p to these structures requires Dnm1p, localization of Mdv1p to mitochondrial membranes does not. This indicates that Mdv1p possesses a Dnm1p-independent mitochondrial targeting signal. Dnm1p-independent targeting of Mdv1p to mitochondria requires MDV2. Our data indicate that MDV2 also functions separately to regulate the assembly of Dnm1p into punctate structures. In contrast, Mdv1p is not required for the assembly of Dnm1p, but Dnm1p-containing punctate structures lacking Mdv1p are not able to complete division. Our studies suggest that mitochondrial fission is a multi-step process in which Mdv2p regulates the assembly of Dnm1p into punctate structures and together with Mdv1p functions later during fission to facilitate Dnm1p-dependent mitochondrial membrane constriction and/or division.     

Mitochondrial fission proteins Fis1 and Mdv1, but not Dnm1, play a role in maintenance of heteroplasmy in budding yeast

In budding yeast, the mitochondrial DNA (mtDNA) replication pathway involving the homologous DNA pairing protein Mhr1 promotes mitochondrial allele segregation. Mitochondrial fusion facilitates the recombination-mediated replication pathway; however, the role of fission remains largely unknown. By monitoring mitochondrial allele segregation during zygotic division, we found that the absence of fission proteins Fis1 or Mdv1, but not Dnm1, resulted in increased initial homoplasmy levels and decreased mtDNA copy number. However, decreases in mtDNA copy number alone were not sufficient for rapid establishment of homoplasmy, suggesting that inhibiting the activities of certain fission proteins promotes homoplasmy by reducing the number of mtDNA segregation units.     

Establishment of stable hFis1 knockdown cells with an siRNA expression vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.     

Analysis of functional domains of rat mitochondrial Fis1, the mitochondrial fission-stimulating protein

In yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (Dnm1p) in conjunction with a peripheral protein, Mdv1p, and a C-tail-anchored outer membrane protein, Fis1p. In mammals, a dynamin-related protein (Drp1) and Fis1 are involved in the mitochondrial-fission reaction as Dnm1 and Fis1 orthologues, respectively. The involvement of other component(s), such as the Mdv1 homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fis1 (rFis1) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFis1 formed a approximately 200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-down by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFis1. These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFis1 interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFis1.     

The conserved fission complex on peroxisomes and mitochondria

Peroxisomes are eukaryotic organelles highly versatile and dynamic in content and abundance. Plant peroxisomes mediate various metabolic pathways, a number of which are completed sequentially in peroxisomes and other subcellular organelles, including mitochondria and chloroplasts. To understand how peroxisomal dynamics contribute to changes in plant physiology and adaptation, the multiplication pathways of peroxisomes are being dissected. Research in Arabidopsis thaliana has identified several evolutionarily conserved families of proteins in peroxisome division. These include five PEROXIN11 proteins (PEX11a to -e) that induce peroxisome elongation and the fission machinery, which is composed of three dynamin-related proteins (DRP3A, -3B and -5B) and DRP''s membrane receptor, FISSION1 (FIS1A and -1B). While the function of PEX11 is restricted to peroxisomes, the fission factors are more promiscuous. DRP3 and FIS1 proteins are shared between peroxisomes and mitochondria, and DRP5B plays a dual role in the division of chloroplasts and peroxisomes. Analysis of the Arabidopsis genome suggests that higher plants may also contain functional homologs of the yeast Mdv1/Caf4 proteins, adaptor proteins that link DRPs to FIS1 on the membrane of both peroxisomes and mitochondria. Sharing a conserved fission machine between these metabolically linked subcellular compartments throughout evolution may have some biological significance.Key words: Arabidopsis, peroxisomal and mitochondrial division, dynamin-related protein (DRP), FISSION1 (FIS1), mitochondrial division 1 (Mdv1), CCR4p-associated factor 4 (Caf4)Peroxisomes are single membrane-delimited organelles involved in a variety of metabolic pathways essential to development.¹ Plant peroxisomes participate in processes such as lipid mobilization, photorespiration, detoxification, hormone biosynthesis and metabolism, and plant-pathogen interaction.^2,3 A number of these metabolic functions, such as photorespiration, fatty acid metabolism and jasmonic acid biosynthesis, are accomplished through the cooperative efforts of peroxisomes and other subcellular compartments, such as mitochondria and chloroplasts.^3–5 The function, morphology and abundance of peroxisomes can vary depending on the organism, cell type, developmental stage and prevailing environmental conditions in which the organism resides.^6,7 It is now believed that in addition to budding from the endoplasmic reticulum (ER), peroxisomes also multiply from pre-existing peroxisomes via division, going through steps including peroxisome elongation/tubulation, membrane constriction and fission.^7,8In the reference plant Arabidopsis thaliana, three evolutionarily conserved families of proteins have been identified as key components of the peroxisome division apparatus. Five integral membrane proteins, named PEX11a to -e, are mainly responsible for inducing the elongation and tubulation of peroxisomes in the early stage of peroxisome division.^9–11 DRP3A and DRP3B are members of a dynamin-related protein family that powers the fission of membranes and FIS1A and FIS1B are homologous proteins believed to anchor the DRP proteins to the membrane.^12–19 Similar to their counterparts in yeasts and mammals, DRP3 and FIS1 are shared by the fission machineries of peroxisomes and mitochondria.^12–19 We recently reported the unexpected finding that DRP5B, a plant/algal-specific DRP distantly related to the DRP3 proteins and originally discovered for its function in chloroplast division, is also involved in the division of peroxisomes. Using co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we further demonstrated that DRP5B and the two DRP3 proteins can homo- and hetero-dimerize and each DRP can form a complex with FIS1A and/or FIS1B and most of the Arabidopsis PEX11 isoforms.²⁰ These results together demonstrate that, despite their distinct evolutionary origins, structures and functions, peroxisomes, mitochondria and chloroplasts use some of the same factors for fission. These data also revealed that, like in yeasts and mammals, the FIS1-DRP complex exits on peroxisomes and mitochondria in plants.DRP5B, a DRP unique in the plant and photosynthetic algae lineages, seems to be the sole component shared by the division of chloroplasts and peroxisomes.²⁰ However, both FIS1 and DRP are found to be required for the division of peroxisomes and mitochondria throughout eukaryotic evolution,^21,22 prompting the question: to what extent is the FIS1-DRP complex conserved among diverse species? In the yeast Saccharomyces cerevisiae, this fission complex also contains an adaptor encoded by two homologous WD40 proteins, Mdv1 and Caf4, which are partially redundant in function with Mdv1 playing the major role. Mdv1 and Caf4 share an N-terminal extension (NTE) domain with two α-helices, a middle coiled-coil domain (CC) and C-terminal WD40 repeat. Both proteins use the NTE to interact with the tetratricopeptide repeat (TPR) domain-containing N-terminus of Fis1, the CC domain to dimerize and the C-terminal WD40 repeat to interact with and recruit the DRP protein, Dnm1.^23,24 The Hansenula polymorpha Mdv1 (Hp Mdv1) also has a dual function in the division of peroxisomes and mitochondria.²⁵ In addition, a Mdv1/Caf4 homolog, Mda1, was identified from the primitive red algae Cyanidioschyzon merolae and found to be involved at least in mitochondrial fission.²⁶ However, higher eukaryotes do not seem to have obvious homologs of Mdv1/Caf4. For example, mammals contain Fis1 and Drp (called DLP1 or Drp1) but no apparent homologs to Mdv1 and Caf4. Instead, a metazoan-specific tail-anchored protein, Mitochondrial Fission Factor (Mff), was recently found to regulate the fission of mitochondria and peroxisomes in a similar manner to Fis1. Mff is essential in recruiting Drp1, at least in mitochondrial division, yet it functions in a Fis1-independent pathway.^27,28To determine whether plants contain structural or functional homologs of Mdv1 and Caf4, we performed blast searches of the Arabidopsis genome, which resulted in the retrieval of ∼300 WD40 proteins. However, just like the search results from mammals, none of these proteins show significant sequence similarity with Mdv1 and Caf4 beyond the WD40 repeats. To identify proteins with similar domain structures with Mdv1/Caf4, we further analyzed these WD40 proteins, using the online Simple Modular Architecture Research Tool (smart.embl-heidelberg.de/). After eliminating proteins apparently inappropriate to be part of this complex, such as kinases and proteins with drastically distinct domain organizations despite of having both WD40 repeats and CC domains, we were able to narrow down to eight proteins. These proteins, which are encoded by At1g04510, At2g32950, At2g33340, At3g18860, At4g05410, At4g21130, At5g50230 and At5g67320, respectively, each contain a central CC domain in addition to the WD40 repeat region and are ranging from 450 to 900 amino acids in length (Fig. 1A). Subcellular localization studies will need to be performed to determine whether some of these proteins are associated with peroxisomes and mitochondria. If such a WD40 protein is proven to be part of the FIS1-DRP complex in Arabidopsis, it will be important to determine whether it simply acts as an adaptor or it also plays other roles, such as to promote and maintain the active structure and conformation of DRP3A/3B at the division site (Fig. 1B). Consistent with the latter scenario, it was found that Sc Mdv1 accumulates at the division sites after Dnm1 assembles and that the mammalian Fis1 and Drp1 proteins physically interact.^29,30 Peroxisomes and mitochondria are functionally linked in a number of metabolic pathways. For example, in plants, they act cooperatively in important processes such as fatty acid metabolism and photorespiration.³ An interesting question to address in the future is whether sharing such a conserved fission machine between peroxisomes and mitochondria throughout evolution has critical biological consequences.Open in a separate windowFigure 1Domain structure of Mdv1/Caf4 and their homologs or putative homologs. (A) Domain structure of Sc Mdv1 and Sc Caf4 from S. cerevisiae, their homologs from H. polymorpha and C. merolae, and the eight Arabidopsis proteins with similar domain organization. Grey boxes indicate the CC domain and black boxes are Wd40 repeats. (B) The putative FIS1-WD40-DRP complex in Arabidopsis. CC, coiled-coil; NTE, N-terminal extension; TPR, tetratricopeptide repeat; TMD, transmembrane domain.