- Email: [email protected]
Role of MINOS in mitochondrial membrane architecture and biogenesis
Opinion
Role of MINOS in mitochondrial membrane architecture and biogenesis Martin van der Laan1,2, Maria Bohnert1, Nils Wiedemann1,2 and Nikolaus Pfanner1,2 1
Institut fu¨r Biochemie und Molekularbiologie, Zentrum fu¨r Biochemie und Molekulare Zellforschung (ZBMZ), Universita¨t Freiburg, 79104 Freiburg, Germany 2 Centre for Biological Signaling Studies (BIOSS), Universita¨t Freiburg, 79104 Freiburg, Germany
Mitochondria possess a complex architecture with two membranes. The inner membrane is divided into two domains: the inner boundary membrane, which is adjacent to the outer membrane, and membrane invaginations termed cristae. Both domains are connected by tubular openings, the crista junctions. Recent studies led to the identification of a large protein complex that is crucial for establishing inner-membrane architecture. This mitochondrial inner-membrane organizing system (MINOS) interacts with protein translocases of the outer membrane that are functionally connected to the endoplasmic reticulum (ER)–mitochondria encounter structure. Here, we propose that MINOS forms a central part of an ER–mitochondria organizing network (ERMIONE) that controls mitochondrial membrane architecture and biogenesis. Mitochondrial membrane architecture Mitochondria are involved in numerous cellular processes. In addition to their central role in ATP production by oxidative phosphorylation, they are involved in the metabolism of amino acids, lipids and iron, and they play important roles in the regulation of programmed cell death (apoptosis) [1–7]. Mitochondria contain two membranes with different functions and architecture. The outer membrane forms the barrier to the rest of the cell. It contains channels formed by the abundant outer-membrane protein porin, also known as the voltage-dependent anion channel (VDAC), which permits the passage of numerous metabolites between cytosol and the mitochondrial intermembrane space. The outer membrane contains also the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM), as well as the sorting and assembly machinery (SAM) and key molecules involved in mitochondrial fusion and fission [1,3,8–12]. The mitochondrial inner membrane possesses a much larger surface than the outer membrane and consists of two topologically different regions: the inner boundary membrane, which is in proximity to the outer membrane, and the cristae membranes that form characteristic membrane invaginations with a large variety of shapes from tubular to lamellar structures [13–17]. The mitochondrial inner membrane is a highly protein-rich membrane, but the proteins are not equally distributed between inner Corresponding author: Pfanner, N. ([email protected]). Keywords: ERMES; MINOS; Mio10; mitochondrial contact sites; mitochondrial cristae; mitofilin.
boundary membrane and cristae membranes, as shown by biochemical fractionation [18] and analysis by electron microscopy and fluorescence microscopy [19–22]. The inner boundary membrane preferentially contains preprotein translocase machineries such as the presequence translocase of the inner membrane (TIM23) and the mitochondrial intermembrane space assembly machinery (Mia40), as well as a fraction of proteins involved in mitochondrial Glossary Crista junctions: the mitochondrial inner membrane consists of the inner boundary membrane (adjacent to the outer membrane) and membrane invaginations (cristae). Crista junctions form the tubular connections between inner boundary membrane and cristae membranes. ERMES: the ER–mitochondria encounter structure (ERMES) physically links the outer mitochondrial membrane with the ER membrane. ERMES is composed of the mitochondrial outer-membrane proteins Mdm10, Mdm34 and Gem1, the ER membrane protein Mmm1 and the adaptor protein Mdm12. ERMIONE: the ER–mitochondria organizing network (ERMIONE) comprises a branched chain of physical and genetic interactions that structurally and functionally link three intracellular membranes and the adjacent aqueous compartments from the mitochondrial matrix to the lumen of the endoplasmic reticulum. Key players in this network are the membrane-bridging ERMES and MINOS complexes. Fusion of mitochondria: fusion of the mitochondrial membranes involves two dynamin-related GTPases, the outer-membrane protein mitofusin/Fzo1, and the inner-membrane protein Opa1/Mgm1. In fungi, the outer-membrane protein Ugo1 functions in an adapter-like manner between Fzo1 and Mgm1. MIA: the mitochondrial intermembrane space import and assembly machinery (MIA) mediates the biogenesis of intermembrane space proteins that contain characteristic cysteine-rich internal signal sequences. This process involves the formation of transient intermolecular disulfides with the intermembrane space receptor Mia40 as well as the activity of the sulfhydryl oxidase Erv1. MINOS: the mitochondrial inner-membrane organizing system (MINOS) is involved in connecting the two subdomains of the inner mitochondrial membrane, the inner boundary membrane and the cristae. In yeast, MINOS consists of at least six different subunits: the mitofilin protein Fcj1, Mio10, Mio27, Aim5, Aim13 and Aim37. MINOS deficiency leads to detachment of cristae from the inner boundary membrane and loss of crista junctions. Additionally, MINOS components are engaged in multiple interactions with the outer mitochondrial membrane. Prohibitins: mitochondrial prohibitins belong to a large protein family also including flotillin and stomatin proteins, characterized by a so-called PHB domain. It has been suggested that these proteins are involved in the organization of membrane domains, most likely through specific lipid-binding properties. SAM: the sorting and assembly machinery (SAM complex) of the outer mitochondrial membrane mediates the membrane integration and folding of b-barrel proteins. SAM is composed of the membrane-integral Sam50 protein and the peripherally associated subunits Sam35 and Sam37. The morphology protein Mdm10 associates with SAM to support the assembly of some a-helical proteins. TOM: the translocase of the outer mitochondrial membrane (TOM complex) mediates the import of the vast majority of nuclear-encoded mitochondrial precursor proteins into the organelle. The protein-conducting channel of the TOM complex is formed by the b-barrel protein Tom40, which associates with several receptor proteins and small subunits involved in stabilization and dynamics of the TOM complex.
0962-8924/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2012.01.004 Trends in Cell Biology, April 2012, Vol. 22, No. 4
185
Opinion
Trends in Cell Biology April 2012, Vol. 22, No. 4
(a) Cytosol Outer membrane Intermembrane space
SAM
Porin
Ugo1
TOM
Mgm1
Mia40
Inner boundary membrane
MINOS
MINOS
TIM23
Crista junction
IV
Cristae membrane
H+
IV
H+
H+
III
H+
H+
III
+
II
Δψ
Mg m1
−
Matrix
III
H+
+
IV
H
III
H+ Fo
H+ Fo
F1
F1
(b)
Wild-type mitochondrion
(c)
MINOS
MitOS
Mitofilin/ Mitofilin/ Fcj1 Fcj1 Mio10
Mos1
MICOS
Yeast ORF name
Fcj1
YKR016W
Mcs10 YCL057C-A
MINOS mutant mitochondrion
MINOS Metazoa
Domain structure 1
+++
1
540
+++
Aim5
Aim5
Mcs12
YBR262C
1
Aim13
Aim13
Mcs19
YFR011C
1
MINOS1
Key:
97
Mitofilin/ IMMT/MINOS2
Signal sequence
106 Transmembrane domain
170
Coiled-coil region
Aim37
Aim37
Mcs27 YNL100W
1
234
Mitofilin domain
Mio27
Mos2
Mcs29
1
233
CHCH motif
CHCHD3/ MINOS3
MOMA-1 YGR235C
TRENDS in Cell Biology
Figure 1. Organization of the mitochondrial inner membrane. (a) Schematic view of mitochondrial outer and inner membranes. The inner membrane consists of two domains – the inner boundary membrane and the cristae membranes – which are connected by crista junctions. Proteins are differentially enriched in the two domains, although the distribution is not static but dynamic. Protein-transport machineries such as the presequence translocase (TIM23) and Mia40 are enriched in the inner boundary membrane, whereas respiratory complexes such as complexes II, III and IV and the F1Fo-ATP synthase are enriched in cristae membranes. Isoforms of the fusion protein Mgm1 are differentially enriched in inner boundary membrane and cristae membrane [97]. The mitochondrial inner-membrane organizing system (MINOS) is enriched in the vicinity of crista junctions. MINOS interacts with protein translocases of the outer membrane (TOM, SAM), the channel-forming protein porin (VDAC), the outer-membrane fusion component Ugo1, and the intermembrane space receptor Mia40. (b) The inner membrane of wild-type mitochondria forms invaginations (cristae). In MINOS mutant mitochondria, most crista junctions are lost and large internal membrane stacks, resembling a labyrinth, are observed. The ancient Greek drachma shows the head of Zeus or his son Minos and the mythical labyrinth of Knossos, Crete; in Greek mythology Minos was a king of Crete when the labyrinth was built. Photo of the drachma with permission from Fritz Rudolf Ku¨nker GmbH & Co. KG, Osnabru¨ck, www.kuenker.de, and Lu¨bke & Wiedemann, Stuttgart. (c) The MINOS complex contains six inner-membrane proteins. The nomenclatures in yeast (left) and metazoa (right) are indicated. ORF, open reading frame and systematic name in the yeast Saccharomyces cerevisiae. The domain structures of the MINOS subunits are shown; numbers refer to the first and last amino acid residues of the yeast proteins.
fusion (Mgm1/Opa1) (Figure 1a). The cristae membranes mainly contain the complexes of the respiratory chain, the F1Fo-ATP synthase, and metabolite carriers such as the ADP/ATP carrier [19,20]. Inner boundary membrane and cristae membranes are connected by tubular openings (Figure 1a,b). These 186
crista junctions (see Glossary) are believed to limit diffusion between both membrane domains, although there is no strict separation, but the distribution of molecules between the membrane domains is dynamic [17,20,22]. Crista junctions are also thought to limit diffusion of soluble molecules between the intracristal space and the remainder of the
Opinion intermembrane space, which is located between inner boundary membrane and outer membrane. Crista junctions thus help to form a microenvironment in the intracristal space that may enhance the capacity of the oxidative phosphorylation system of mitochondria [16,17,23–25]. Despite detailed morphological analyses of mitochondrial ultrastructure, little has been known about the molecules that are involved in the maintenance of cristae organization [16,17]. Recent studies by several groups led to the identification of a large protein complex that functions as a mitochondrial inner-membrane organizing system (MINOS) [26–29]. Surprisingly, MINOS interacts with a variety of different partner proteins, including several protein complexes of the mitochondrial outer membrane (Figure 1a). Here, we put the different observations into context and propose that MINOS forms a central core of a large organizing system that extends from ER–mitochondria junctions to mitochondrial outer and inner membranes and mitochondrial DNA (mtDNA) nucleoids. We term this organizing system ERMIONE, for ER–mitochondria organizing network, and suggest that ERMIONE functions as a regulatory system that includes both biochemical and genetic interactions. Mitochondrial inner-membrane organization Yeast mutants that affect the dimerization of the F1Fo-ATP synthase display altered cristae morphologies [23,29–33]. The F1Fo-ATP synthase can assemble into large spiral-like oligomeric structures that may form a scaffold for the stabilization of cristae structures, in particular for the formation of cristae tips [24,25,33,34]. However, the F1Fo-ATP synthase is probably not responsible for the formation or maintenance of crista junctions [33]. The inner-membrane protein Mgm1 (yeast)/Opa1 (mammals) is required for fusion of mitochondrial membranes [35–39]. In addition, mutants of Mgm1/Opa1 show altered cristae morphology. It is thought that oligomerization of Mgm1/Opa1 is required
Trends in Cell Biology April 2012, Vol. 22, No. 4
for normal cristae structure [37,39,40], although further work is needed to define the molecular mechanisms. The inner-membrane protein Mdm33 also affects innermembrane structure, possibly by regulating innermembrane division [41]. Characterization of the mitochondrial inner-membrane protein mitofilin was a major step in the analysis of crista junctions [26–29,33,42–44]. Mitofilin was initially identified in heart muscle cells [45] and received its name due to its colocalization with the filamentous mitochondrial network in fibroblasts [46]. Mitofilin was found in the proteome of purified mitochondria [47,48]. It is targeted to mitochondria by an N-terminal presequence and anchored in the inner membrane by a single hydrophobic transmembrane region that follows the presequence. The remainder of the protein is exposed to the mitochondrial intermembrane space and includes a coiled-coil region that supports protein–protein interactions (Figure 1c) [33,42]. Cells with decreased levels of mitofilin or mutant cells completely lacking mitofilin show a striking alteration of the inner morphology of mitochondria. The cristae membranes are detached from the inner boundary membrane and form large internal membrane stacks (Figure 1b) [33,42]. Because crista junctions are largely absent in mitofilin mutant cells, it was concluded that mitofilin is required for formation of crista junctions, and was therefore also termed Fcj1 in yeast [33]. However, crista junctions are not completely absent in mitofilin mutants [26,28] and thus mitofilin is not strictly essential for their formation. The available results are explained best by a role of mitofilin in the maintenance of crista junctions, including the prevention of cristae release from the inner boundary membrane. Though it was originally assumed that mitofilin mainly undergoes homotypic interactions [33,42], recent studies have led to the identification of a large hetero-oligomeric protein complex in the inner membrane that contains
Box 1. Identification of the mitochondrial inner-membrane organizing system Four independent studies identified a large protein complex of the inner mitochondrial membrane that is crucial for maintenance of the typical inner-membrane morphology. The complex consists of the core components mitofilin/Fcj1 and MINOS1/Mio10 together with at least four additional subunits. The complex has received three different names: MINOS, MICOS, and MitOS. All studies concordantly report that mutations affecting this protein complex induce a dramatic loss of crista junctions and a detachment of cristae membranes from the inner boundary membrane. von der Malsburg et al. [26] combined stable isotope-labeling with amino acids in cell culture (SILAC) with purification of mitofilincontaining protein complexes by affinity chromatography and mass spectrometric analysis. The identified protein complex is composed of mitofilin/Fcj1, Mio10, Mio27, Aim5, Aim13 and Aim37 and was termed mitochondrial inner-membrane organizing system (MINOS). This study also identified a physical interaction between mitofilin/ Fcj1 and the outer-membrane TOM complex. This interaction is important for protein import into the intermembrane space via the MIA pathway. Harner et al. [27] expressed a synthetic linker protein in yeast cells as a marker for contact sites between outer and inner mitochondrial membranes. They fractionated mitochondrial membranes and analyzed proteins enriched in contact-site fractions by mass spectrometry. Six of the proteins found in contact-site fractions were shown to form an inner-membrane complex preferentially
located at crista junctions, named the mitochondrial contact-site (MICOS) complex. This study provided evidence for interaction of this complex with the Ugo1 fusion component of the outer membrane and for an interaction with the SAM complex (in agreement with further studies [51–53]). Hoppins et al. [28] systematically analyzed genes involved in mitochondrial biogenesis and function for similar synthetic interaction patterns (MITO-MAP). One gene cluster including FCJ1 encoded subunits of a protein complex required for maintenance of mitochondrial inner-membrane morphology, termed mitochondrial organizing structure (MitOS). MitOS genes showed strong genetic interactions with ERMES and phospholipid biosynthesis genes. Fluorescence microscopy analysis of cells expressing GFP-tagged subunits suggested the formation of large structures spanning the mitochondrial intermembrane space. Alkhaja et al. [29] identified Mio10 as a protein potentially stabilizing mitochondrial membrane protein supercomplexes. Tagged Mio10 co-purified mitofilin/Fcj1 and further components of MINOS. The human ortholog of Mio10 is also part of a mitofilincontaining complex and was termed MINOS1. Further components are the CHCHD3/MINOS3 protein, which belongs to the same protein family as yeast Aim13, as well as HSPA9 and the J-protein DnaJC11. In agreement with further studies [51–53], this work demonstrated a direct interaction of the MINOS complex with the SAM complex of the outer mitochondrial membrane. 187
Opinion
Trends in Cell Biology April 2012, Vol. 22, No. 4
mitofilin as one of the core components (Figure 1a,c; Box 1) [26–29]. This large mitofilin complex was termed mitochondrial inner-membrane organizing system (MINOS) [26,29]. It is a remarkable coincidence that MINOS was identified in parallel by four independent studies. Thus the complex received the further names MICOS (mitochondrial contact site) and MitOS (mitochondrial organizing structure) [27,28]. Different experimental strategies and approaches led to the identification of MINOS/MICOS/MitOS (Box 1) [26–29]. The MINOS complex contains six different subunits (Figures 1c, 2). Mitofilin/Fcj1 and the small integral inner-membrane protein Mio10 are the two core components [26–29]. The absence of either leads to dissociation of the MINOS complex and massive rearrangement of inner-membrane morphology, with the loss of crista junctions and the appearance of large internal membrane stacks. Three MINOS subunits were originally identified in a genetic screen for proteins that are involved in the inheritance of mtDNA,
and were termed Aim5, Aim13 and Aim37 (altered inheritance of mitochondria) (currently, more than 40 AIM genes are listed in the Saccharomyces genome database, although the molecular function of many of them is not yet known) [49]. The three Aim proteins of the MINOS complex are all inner-membrane proteins, as are mitofilin and Mio10. Aim5 and Aim37 are integral membrane proteins, whereas Aim13 is a peripheral membrane protein (Figure 1c). Mutants of these AIM genes show phenotypes that are in principle similar to those of mutants lacking mitofilin or Mio10; however, the defects are not as severe. Lack of Aim proteins leads to a partial dissociation of the MINOS complex, a reduction of the number of crista junctions, and a partial alteration of mitochondrial morphology (with internal cristae stacks) [26–28]. The sixth subunit of the MINOS complex, Mio27, is related to Aim37; Mio27 mutants show only mild phenotypes. The recently identified C. elegans mitochondrial morphology protein MOMA-1 is an ortholog of Aim37 and Mio27 [44] (Figure 1c).
Endoplasmic reticulum
Cytosol
Mitochondrion
ER lumen
ER membrane Cytosol OM IMS IM
Gem Mmm 1 12 1
Lipid transport
34 Mdm 10
Prohibitins
Mfn 2 Mfn 1/2
ERMES Tom7 Mdm 10
Mdm 10 SAM
13 Fcj1 5Aim 37 10 27 Mio
Mdm 31 32
MINOS complexes
Matrix
Porin
Crista junction
Ca++ flux
TOM Opa1/ Mgm1
Mia40 TIM23
MINOS complexes
Nucleoid TRENDS in Cell Biology
Figure 2. An ER–mitochondria organizing network (ERMIONE). The ER and the mitochondrial outer membrane, as well as the mitochondrial outer and inner membranes, are connected by protein–protein interactions. The lower part of the figure shows a schematic view of protein complexes that are involved in membrane contact sites. Biochemically demonstrated interactions are indicated by double arrows; genetic interactions are indicated by dotted double arrows. ER and mitochondria can be physically connected by the ERMES complex and by mitofusins (Mfn). MINOS complexes probably form an extended network on the intermembrane space side of the inner membrane. MINOS interacts with several outer-membrane proteins, including the protein translocases TOM and SAM, the abundant channel-protein porin, and fusion components. MINOS transiently interacts with Mia40 and promotes protein import through TOM into the intermembrane space. MINOS and ERMES have been linked genetically by analysis of double mutants and biochemically via the SAM complex (SAM interacts with MINOS, and the morphology protein Mdm10 is present in both SAM and ERMES). ERMES, MINOS and further inner-membrane proteins, including prohibitins and Mdm31/32, are involved in the maintenance of mitochondrial DNA that is organized in nucleoids. The network of ER–mitochondrial outer-membrane–inner-membrane interactions is termed the ER–mitochondria organizing network (ERMIONE). This network of interactions promotes the transport of lipids and calcium ions between the compartments. OM, outer membrane; IMS, intermembrane space; IM, inner membrane.
188
Opinion The MINOS complex forms a large structure in the megadalton range and thus its subunits are probably present in multiple copies (Figure 2). Mitofilin and Mio10 contain sequence elements that favor protein– protein interactions and probably form homo- and hetero-oligomeric structures. Affinity-purification studies yielded various combinations of MINOS components, suggesting that multiple subcomplexes may exist that are arranged in a large, dynamic superstructure [26–29]. MINOS components are enriched in the vicinity of crista junctions [27,33], and all subunits contain domains that are exposed to the intermembrane space. Taking the biochemical and morphological evidence together [26–29], MINOS may form an extended network on the intermembrane space side of the inner membrane that is important for the maintenance of inner-membrane organization. The formation of the typical cristae structures probably involves several dynamic and structural elements, including MINOS, the F1Fo-ATP synthase, and Mgm1/Opa1. Multiple interactions of MINOS with the mitochondrial outer membrane Interactions of the MINOS complex are not limited to the mitochondrial inner membrane. Several studies surprisingly demonstrated that proteins and protein complexes of the mitochondrial outer membrane physically interact with MINOS components. The outer membrane contains two major machineries for protein transport – the TOM complex that is responsible for importing hundreds of different nuclear-encoded proteins into mitochondria, and the SAM complex (TOB complex) that mediates the insertion of b-barrel proteins into the outer membrane [1,8,9,50]. Both translocase machineries were found as interaction partners of MINOS (Figures 1a,2) [26–29, 51–53]. In the case of the TOM complex, MINOS indeed plays a supportive role in protein import. However, MINOS does not promote the import of precursor proteins into or across the inner membrane; the presequence translocase (TIM23 complex) and the carrier translocase (TIM22 complex) of the inner membrane have not been found in association with MINOS. Instead, MINOS transiently interacts with the intermembrane space import receptor Mia40 [26]. Mia40 is the core of the mitochondrial intermembrane-space assembly machinery that drives the import of proteins into the intermembrane space by an oxidative mechanism [9,54–57]. Mia40 directly interacts with the incoming substrate proteins via disulfide bonds. To capture immediately preproteins in transit through the TOM channel, Mia40 binds to the signal sequences of incoming preproteins as soon as they emerge on the intermembrane space side of the TOM complex [26]. For this task, Mia40 has to be positioned in close vicinity to the TOM complex, and this is achieved by the transient interaction of mitofilin with both the TOM complex and Mia40. Mitofilin may thus perform adapter-like functions to link different machineries of the mitochondrial protein-import apparatus. The exact molecular function of the interaction of MINOS with the SAM complex has not yet been defined. An adapter/organizing role of MINOS may support the cooperation of TOM and SAM, and thus MINOS may assist in the transfer of b-barrel proteins from the TOM complex
Trends in Cell Biology April 2012, Vol. 22, No. 4
to the SAM complex. In cells with reduced levels of Sam50, the cristae architecture was altered [53], suggesting that the interaction of MINOS with the outer membrane may be important for maintaining inner-membrane architecture. In addition to the association with protein translocases, MINOS was also found to interact with the most abundant outer-membrane protein porin (VDAC), as well as with the fusion component Ugo1 (Figures 1a, 2) [27,28]. Porin is the major channel for transport of metabolites between the cytosol and the intermembrane space. Because metabolites are differentially distributed between intracristal space and the remainder of the intermembrane space [14,16, 58], the enrichment of MINOS in the vicinity of crista junctions may help to position the main metabolite channel of mitochondria close to crista junctions, and may thus facilitate the efficient distribution of metabolites. Ugo1 is a subunit of the outer-membrane fusion machinery of yeast mitochondria and has an adapter-like function through its linkage of two dynamin-like GTPases: the outermembrane protein Fzo1 (termed mitofusin in higher eukaryotes) and Mgm1/Opa1 of the inner membrane [59–61]. Although it is an open question whether MINOS is present in a supercomplex with Fzo1 and Mgm1, or if MINOS interacts with Ugo1 only [26,27,52], the interaction of MINOS with Ugo1 provides a link between two machineries that control mitochondrial membrane morphology and dynamics. An ER–mitochondria organizing network The large number of reported MINOS interaction partners of seemingly unrelated functions raises the question of how these findings can be put into a functional context. We propose that MINOS forms a central core of a large organizing network of mitochondria that includes numerous further components. The large size of MINOS may provide a scaffold for multiple binding partners and thus help to spatially organize and coordinate mitochondrial functions (Figure 2). Of particular importance is the functional connection of SAM and TOM to the ER–mitochondria encounter structure (ERMES) [62–65]. In fungi, ERMES physically connects ER and mitochondria by containing both an ER-located subunit (Mmm1) and mitochondrial-located subunits within the same complex (Figure 2). Remarkably, the morphology protein Mdm10 of the mitochondrial outer membrane has dual localization in SAM and ERMES [66–70]. A small TOM subunit also exhibits dual localization: Tom7 is present in the TOM complex, and also associates with Mdm10, and thus regulates the distribution of Mdm10 between SAM and ERMES [67,69,71]. Yeast mutants of ERMES components show a dramatic alteration of mitochondrial shape, leading to giant ball-like mitochondrial structures [67,68,72–79]. Yeast mutants of SAM components as well as of several TOM components lead to a similar phenotype [66,67], underscoring the functional connection between ERMES, SAM and TOM. Remarkably, mutants of the ER-located subunit Mmm1 were also reported to affect the ultrastructure of the mitochondrial inner membrane, leading to a massive disorganization of cristae membranes [80], suggesting an important connection from the ER to the mitochondrial 189
Opinion inner membrane. Indeed, strong genetic interactions have been observed between ERMES and MINOS components [28]. Taken together, we propose that ERMES, SAM, TOM and MINOS are connected in a dynamic network and thus link ER to mitochondrial outer and inner membranes. ERMES as well as MINOS have been implicated in lipid transfer and metabolism [2,27,28,62–65,81] and thus they may provide a scaffold for transport of lipid molecules between ER and both mitochondrial membranes [82]. Both complexes are genetically linked to the prohibitin ring complexes of the inner membrane that are integrated into a network of mitochondrial lipid metabolism [2,28,81]. In addition, prohibitins affect cristae morphogenesis by regulating the processing of the fusion protein Mgm1/ Opa1 [83]. The close connections between ER and mitochondria probably facilitate the uptake of calcium ions by mitochondria upon their release from the ER [84]. In higher eukaryotes, mitofusins have been reported to link physically ER and mitochondria and thus to promote calcium transfer into mitochondria (Figure 2) [85]. It is an open question whether ERMES and mitofusin tethers exist in parallel in the same organism. Interestingly, in yeast MINOS interacts with the fusion component Ugo1 that binds to Fzo1, the yeast ortholog of mitofusins [27,59]. Although an ortholog of Ugo1 has not yet been found in higher eukaryotes, it is tempting to speculate that MINOS, whose core components are conserved from yeast to human (Figure 1c), is the connecting partner of both systems that link mitochondria and the ER, providing a communication chain across three organellar membranes. ERMES has also been proposed to be involved in mitochondrial distribution and the connection of mitochondria to the cytoskeleton [73–75], although different views exist on the molecular mechanism. The recent identification of the GTPase Gem1 as a mitochondria-bound subunit of ERMES [86,87] provides an interesting twist because the protein Miro (mitochondrial Rho-like), the homolog of Gem1 in higher eukaryotes, functions as an adapter in linking mitochondria to the cytoskeleton [88–90]. Four of the MINOS subunits were found in a genetic screen for altered inheritance of mitochondria, reflected in the names Aim5, Aim13 and Aim37; the fourth, mitofilin/ Fcj1, was also termed Aim28 [49]. MINOS is thus involved in the maintenance of mtDNA. Yeast mtDNA is organized into DNA–protein complexes termed nucleoids, and ERMES can be found in foci that are adjacent to a subset of nucleoids [75,78,80,91]. Genetic interaction studies revealed a functional link between ERMES, MINOS and further inner-membrane proteins that are involved in regulating mitochondrial morphology and the maintenance of mtDNA. These include the morphology proteins Mdm31 and Mdm32 that are anchored in the inner membrane and expose large domains to the intermembrane space [79]. Yeast mutants of Mdm31 and Mdm32 show a similar alteration of mitochondrial shape as ERMES, SAM and TOM mutants (i.e. large spherical mitochondria), and Mdm31 and Mdm32 are required to organize mtDNA into nucleoids [79]. It is thus likely that MINOS, ERMES and further Mdm proteins functionally cooperate in the maintenance and organization of mtDNA (Figure 2). 190
Trends in Cell Biology April 2012, Vol. 22, No. 4
Box 2. Outstanding questions What is the mechanistic role of MINOS in the formation and maintenance of crista junctions? What are the physiological consequences of the segregation of cristae tubules from the inner boundary membrane in MINOS mutants? How dynamic are the ERMES and MINOS complexes and which factors regulate their assembly state? Do ERMES and MINOS directly interact with each other or are further components involved? How are the different phenotypes associated with mutations of ERMIONE components, including membrane morphology, lipid metabolism, protein biogenesis and mtDNA maintenance, related to each other at the molecular level? Are subcomplexes of MINOS or ERMES responsible for distinct functions? Are MINOS or ERMES involved in mitochondrial or ER functions during programmed cell death? Is there a signaling pathway extending from the mitochondrial matrix via MINOS and ERMES to the ER lumen and eventually to the nucleus?
Taken together, we propose that ERMES and MINOS are linked into an ER–mitochondria organizing network (ERMIONE) that includes machineries for protein biogenesis, lipid metabolism, metabolite and ion transport, and mtDNA organization. ER–mitochondria interaction sites have also been shown to be related to sites of mitochondrial division [92] although the molecular mechanisms are not yet known. Cristae remodeling can occur during mitochondrial steps of apoptosis [16,40,93–96]. Future studies will need to address whether MINOS, ERMES and further components of ERMIONE play a role in mitochondrial fission and apoptotic events. Concluding remarks Morphological and genetic studies led to the suggestion that there could be a multi-membrane-spanning connection from ERMES across the mitochondrial membranes to mtDNA nucleoids [63,64,75,76,79,80,91]. However, only limited information was available on the molecular composition and structural arrangement of such an organizing center. The identification of MINOS and its multiple interactions provide a new view on mitochondrial architecture and biogenesis. MINOS forms a large scaffold and interaction platform that connects to outer-membrane complexes, including ERMES, as well as to machineries inside mitochondria. The hypothesis of a large ER–mitochondria organizing network (ERMIONE) will provide a platform for research on the integrative organization of mitochondrial functions and a number of exciting questions for future research remain (Box 2). Acknowledgments This work has been supported by the Deutsche Forschungsgemeinschaft, the Excellence Initiative of the German Federal & State Governments (EXC 294), the Gottfried Wilhelm Leibniz Program, Sonderforschungsbereich 746, Landesforschungspreis Baden-Wu¨rttemberg, and the Bundesministerium fu¨r Bildung und Forschung.
References 1 Dolezal, P. et al. (2006) Evolution of the molecular machines for protein import into mitochondria. Science 313, 314–318 2 Osman, C. et al. (2011) Making heads or tails of phospholipids in mitochondria. J. Cell Biol. 192, 7–16
Opinion 3 Otera, H. and Mihara, K. (2011) Molecular mechanisms and physiologic functions of mitochondrial dynamics. J. Biochem. 149, 241–251 4 Scheffler, I.E. (2008) Mitochondria (2nd edn), J. Wiley and Sons 5 McBride, H.M. et al. (2006) Mitochondria: more than just a powerhouse. Curr. Biol. 16, R551–R560 6 Wang, C. and Youle, R.J. (2009) The role of mitochondria in apoptosis. Annu. Rev. Genet. 43, 95–118 7 Lill, R. (2009) Function and biogenesis of iron–sulphur proteins. Nature 460, 831–838 8 Neupert, W. and Herrmann, J.M. (2007) Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 9 Chacinska, A. et al. (2009) Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644 10 Okamoto, K. and Shaw, J.M. (2005) Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu. Rev. Genet. 39, 503–536 11 Westermann, B. (2010) Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell Biol. 11, 872–884 12 Palmer, C.S. et al. (2011) The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery. Cell Signal. 23, 1534–1545 13 Perkins, G. et al. (1997) Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts. J. Struct. Biol. 119, 260–272 14 Frey, T.G. and Mannella, C.A. (2000) The internal structure of mitochondria. Trends Biochem. Sci. 25, 319–324 15 Frey, T.G. et al. (2002) Insight into mitochondrial structure and function from electron tomography. Biochim. Biophys. Acta 1555, 196–203 16 Mannella, C.A. (2006) The relevance of mitochondrial membrane topology to mitochondrial function. Biochim. Biophys. Acta 1762, 140–147 17 Zick, M. et al. (2009) Cristae formation – linking ultrastructure and function of mitochondria. Biochim. Biophys. Acta 1793, 5–19 18 Werner, S. and Neupert, W. (1972) Functional and biogenetical heterogeneity of the inner membrane of rat-liver mitochondria. Eur. J. Biochem. 25, 379–396 19 Gilkerson, R.W. et al. (2003) The cristal membrane of mitochondria is the principal site of oxidative phosphorylation. FEBS Lett. 546, 355–358 20 Vogel, F. et al. (2006) Dynamic subcompartmentalization of the mitochondrial inner membrane. J. Cell Biol. 175, 237–247 21 Wurm, C.A. and Jakobs, S. (2006) Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast. FEBS Lett. 580, 5628–5634 22 Suppanz, I.E. et al. (2009) The m-AAA protease processes cytochrome c peroxidase preferentially at the inner boundary membrane of mitochondria. Mol. Biol. Cell 20, 572–580 23 Bornho¨vd, C. et al. (2006) Mitochondrial membrane potential is dependent on the oligomeric state of F1Fo-ATP synthase supracomplexes. J. Biol. Chem. 281, 13990–13998 24 Strauss, M. et al. (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J. 27, 1154–1160 25 Davies, K.M. et al. (2011) Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc. Natl. Acad. Sci. U.S.A. 108, 14121–14126 26 von der Malsburg, K. et al. (2011) Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis. Dev. Cell 21, 694–707 27 Harner, M. et al. (2011) The mitochondrial contact site complex, a determinant of mitochondrial architecture. EMBO J. 30, 4356–4370 28 Hoppins, S. et al. (2011) A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria. J. Cell Biol. 195, 323–340 29 Alkhaja, A.K. et al. (2012) MINOS1 is a conserved component of mitofilin complexes and required for mitochondrial function and cristae organization. Mol. Biol. Cell 23, 247–257 30 Giraud, M.F. et al. (2002) Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? Biochim. Biophys. Acta 1555, 174–180 31 Paumard, P. et al. (2002) The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J. 21, 221–230 32 Arselin, G. et al. (2004) The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology. J. Biol. Chem. 279, 40392–40399
Trends in Cell Biology April 2012, Vol. 22, No. 4
33 Rabl, R. et al. (2009) Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g. J. Cell Biol. 185, 1047–1063 34 Buzhynskyy, N. et al. (2007) Rows of ATP synthase dimers in native mitochondrial inner membranes. Biophys. J. 93, 2870–2876 35 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 36 Cipolat, S. et al. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U.S.A. 101, 15927–15932 37 Meeusen, S. et al. (2006) Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Cell 127, 383–395 38 Ishihara, N. et al. (2006) Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. EMBO J. 25, 2966–2977 39 DeVay, R.M. et al. (2009) Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion. J. Cell Biol. 186, 793–803 40 Frezza, C. et al. (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126, 177–189 41 Messerschmitt, M. et al. (2003) The inner membrane protein Mdm33 controls mitochondrial morphology in yeast. J. Cell Biol. 160, 553–564 42 John, G.B. et al. (2005) The mitochondrial inner membrane protein mitofilin controls cristae morphology. Mol. Biol. Cell 16, 1543–1554 43 Mun, J.Y. et al. (2010) Caenorhabditis elegans mitofilin homologs control the morphology of mitochondrial cristae and influence reproduction and physiology. J. Cell. Physiol. 224, 748–756 44 Head, B.P. et al. (2011) A novel mitochondrial outer membrane protein, MOMA-1, that affects cristae morphology in Caenorhabditis elegans. Mol. Biol. Cell 22, 831–841 45 Icho, T. et al. (1994) A novel human gene that is preferentially transcribed in heart muscle. Gene 144, 301–306 46 Odgren, P.R. et al. (1996) Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains. J. Cell Sci. 109, 2253–2264 47 Sickmann, A. et al. (2003) The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U.S.A. 100, 13207–13212 48 Taylor, S.W. et al. (2003) Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 21, 281–286 49 Hess, D.C. et al. (2009) Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis. PLoS Genet. 5, e1000407 50 Dukanovic, J. and Rapaport, D. (2011) Multiple pathways in the integration of proteins into the mitochondrial outer membrane. Biochim. Biophys. Acta 1808, 971–980 51 Xie, J. et al. (2007) The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiledcoil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11. FEBS Lett. 581, 3545–3549 52 Darshi, M. et al. (2011) ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function. J. Biol. Chem. 286, 2918–2932 53 Ott, C. et al. (2012) Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes. Mol. Cell. Biol. 32, 1173–1188 54 Koehler, C.M. and Tienson, H.L. (2009) Redox regulation of protein folding in the mitochondrial intermembrane space. Biochim. Biophys. Acta 1793, 139–145 55 Herrmann, J.M. and Riemer, J. (2012) The mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space. J. Biol. Chem. 287, 4426–4433 56 Sideris, D.P. and Tokatlidis, K. (2010) Oxidative protein folding in the mitochondrial intermembrane space. Antioxid. Redox Signal. 13, 1189–1204 57 Endo, T. et al. (2010) Structural basis for the disulfide relay system in the mitochondrial intermembrane space. Antioxid. Redox Signal. 13, 1359–1373 58 Sukhorukov, V.M. and Bereiter-Hahn, J. (2009) Anomalous diffusion induced by cristae geometry in the inner mitochondrial membrane. PLoS ONE 4, e4604 59 Sesaki, H. and Jensen, R.E. (2004) Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion. J. Biol. Chem. 279, 28298–28303 191
Opinion 60 Hoppins, S. et al. (2009) Mitochondrial outer and inner membrane fusion requires a modified carrier protein. J. Cell Biol. 184, 569–581 61 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 62 Kornmann, B. et al. (2009) An ER–mitochondrial tethering complex revealed by a synthetic biology screen. Science 325, 477–481 63 Wiedemann, N. et al. (2009) Cell biology: connecting organelles. Science 325, 403–404 64 Kornmann, B. and Walter, P. (2010) ERMES-mediated ER– mitochondria contacts: molecular hubs for the regulation of mitochondrial biology. J. Cell Sci. 123, 1389–1393 65 Elbaz, Y. and Schuldiner, M. (2011) Staying in touch: the molecular era of organelle contact sites. Trends Biochem. Sci. 36, 616–623 66 Meisinger, C. et al. (2004) The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane. Dev. Cell 7, 61–71 67 Meisinger, C. et al. (2006) Mitochondrial protein sorting: differentiation of b-barrel assembly by Tom7-mediated segregation of Mdm10. J. Biol. Chem. 281, 22819–22826 68 Meisinger, C. et al. (2007) The morphology proteins Mdm12/Mmm1 function in the major b-barrel assembly pathway of mitochondria. EMBO J. 26, 2229–2239 69 Yamano, K. et al. (2010) Tom7 regulates Mdm10-mediated assembly of the mitochondrial import channel protein Tom40. J. Biol. Chem. 285, 41222–41231 70 Wideman, J.G. et al. (2010) Roles of the Mdm10, Tom7, Mdm12, and Mmm1 proteins in the assembly of mitochondrial outer membrane proteins in Neurospora crassa. Mol. Biol. Cell 21, 1725–1736 71 Becker, T. et al. (2011) Biogenesis of mitochondria: dual role of Tom7 in modulating assembly of the preprotein translocase of the outer membrane. J. Mol. Biol. 405, 113–124 72 Burgess, S.M. et al. (1994) MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria. J. Cell Biol. 126, 1375–1391 73 Sogo, L.F. and Yaffe, M.P. (1994) Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane. J. Cell Biol. 126, 1361–1373 74 Berger, K.H. et al. (1997) Mdm12p, a component required for mitochondrial inheritance that is conserved between budding and fission yeast. J. Cell Biol. 136, 545–553 75 Boldogh, I.R. et al. (2003) A protein complex containing Mdm10p, Mdm12p, and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery. Mol. Biol. Cell 14, 4618–4627 76 Kondo-Okamoto, N. et al. (2003) Mmm1p spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation. J. Biol. Chem. 278, 48997–49005 77 Frederick, R.L. et al. (2004) Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway. J. Cell Biol. 167, 87–98 78 Youngman, M.J. et al. (2004) Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids. J. Cell Biol. 164, 677–688
192
Trends in Cell Biology April 2012, Vol. 22, No. 4
79 Dimmer, K.S. et al. (2005) Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast. J. Cell Biol. 168, 103–115 80 Hobbs, A.E. et al. (2001) Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability. J. Cell Biol. 152, 401–410 81 Osman, C. et al. (2009) The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria. J. Cell Biol. 184, 583–596 82 Achleitner, G. et al. (1999) Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur. J. Biochem. 264, 545–553 83 Merkwirth, C. et al. (2008) Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria. Genes Dev. 22, 476–488 84 Szabadkai, G. et al. (2006) Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J. Cell Biol. 175, 901–911 85 de Brito, O.M. and Scorrano, L. (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456, 605–610 86 Kornmann, B. et al. (2011) The conserved GTPase Gem1 regulates endoplasmic reticulum–mitochondria connections. Proc. Natl. Acad. Sci. U.S.A. 108, 14151–14156 87 Stroud, D.A. et al. (2011) Composition and topology of the endoplasmic reticulum-mitochondria encounter structure. J. Mol. Biol. 413, 743–750 88 Glater, E.E. et al. (2006) Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent. J. Cell Biol. 173, 545–557 89 Wang, X. and Schwarz, T.L. (2009) The mechanism of Ca2+-dependent regulation of kinesin-mediated mitochondrial motility. Cell 136, 163–174 90 Koshiba, T. et al. (2011) Structure–function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance. J. Biol. Chem. 286, 354–362 91 Meeusen, S. and Nunnari, J. (2003) Evidence for a two membranespanning autonomous mitochondrial DNA replisome. J. Cell Biol. 163, 503–510 92 Friedman, J.R. et al. (2011) ER tubules mark sites of mitochondrial division. Science 334, 358–362 93 Scorrano, L. et al. (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell 2, 55–67 94 Karbowski, M. and Youle, R.J. (2003) Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ. 10, 870–880 95 Germain, M. et al. (2005) Endoplasmic reticulum BIK initiates DRP1regulated remodelling of mitochondrial cristae during apoptosis. EMBO J. 24, 1546–1556 96 Yamaguchi, R. et al. (2008) Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization. Mol. Cell 31, 557–569 97 Zick, M. et al. (2009) Distinct roles of the two isoforms of the dynamin-like GTPase Mgm1 in mitochondrial fusion. FEBS Lett. 583, 2237–2243
Role of MINOS in mitochondrial membrane architecture and biogenesis Martin van der Laan1,2, Maria Bohnert1, Nils Wiedemann1,2 and Nikolaus Pfanner1,2 1
Institut fu¨r Biochemie und Molekularbiologie, Zentrum fu¨r Biochemie und Molekulare Zellforschung (ZBMZ), Universita¨t Freiburg, 79104 Freiburg, Germany 2 Centre for Biological Signaling Studies (BIOSS), Universita¨t Freiburg, 79104 Freiburg, Germany
Mitochondria possess a complex architecture with two membranes. The inner membrane is divided into two domains: the inner boundary membrane, which is adjacent to the outer membrane, and membrane invaginations termed cristae. Both domains are connected by tubular openings, the crista junctions. Recent studies led to the identification of a large protein complex that is crucial for establishing inner-membrane architecture. This mitochondrial inner-membrane organizing system (MINOS) interacts with protein translocases of the outer membrane that are functionally connected to the endoplasmic reticulum (ER)–mitochondria encounter structure. Here, we propose that MINOS forms a central part of an ER–mitochondria organizing network (ERMIONE) that controls mitochondrial membrane architecture and biogenesis. Mitochondrial membrane architecture Mitochondria are involved in numerous cellular processes. In addition to their central role in ATP production by oxidative phosphorylation, they are involved in the metabolism of amino acids, lipids and iron, and they play important roles in the regulation of programmed cell death (apoptosis) [1–7]. Mitochondria contain two membranes with different functions and architecture. The outer membrane forms the barrier to the rest of the cell. It contains channels formed by the abundant outer-membrane protein porin, also known as the voltage-dependent anion channel (VDAC), which permits the passage of numerous metabolites between cytosol and the mitochondrial intermembrane space. The outer membrane contains also the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM), as well as the sorting and assembly machinery (SAM) and key molecules involved in mitochondrial fusion and fission [1,3,8–12]. The mitochondrial inner membrane possesses a much larger surface than the outer membrane and consists of two topologically different regions: the inner boundary membrane, which is in proximity to the outer membrane, and the cristae membranes that form characteristic membrane invaginations with a large variety of shapes from tubular to lamellar structures [13–17]. The mitochondrial inner membrane is a highly protein-rich membrane, but the proteins are not equally distributed between inner Corresponding author: Pfanner, N. ([email protected]). Keywords: ERMES; MINOS; Mio10; mitochondrial contact sites; mitochondrial cristae; mitofilin.
boundary membrane and cristae membranes, as shown by biochemical fractionation [18] and analysis by electron microscopy and fluorescence microscopy [19–22]. The inner boundary membrane preferentially contains preprotein translocase machineries such as the presequence translocase of the inner membrane (TIM23) and the mitochondrial intermembrane space assembly machinery (Mia40), as well as a fraction of proteins involved in mitochondrial Glossary Crista junctions: the mitochondrial inner membrane consists of the inner boundary membrane (adjacent to the outer membrane) and membrane invaginations (cristae). Crista junctions form the tubular connections between inner boundary membrane and cristae membranes. ERMES: the ER–mitochondria encounter structure (ERMES) physically links the outer mitochondrial membrane with the ER membrane. ERMES is composed of the mitochondrial outer-membrane proteins Mdm10, Mdm34 and Gem1, the ER membrane protein Mmm1 and the adaptor protein Mdm12. ERMIONE: the ER–mitochondria organizing network (ERMIONE) comprises a branched chain of physical and genetic interactions that structurally and functionally link three intracellular membranes and the adjacent aqueous compartments from the mitochondrial matrix to the lumen of the endoplasmic reticulum. Key players in this network are the membrane-bridging ERMES and MINOS complexes. Fusion of mitochondria: fusion of the mitochondrial membranes involves two dynamin-related GTPases, the outer-membrane protein mitofusin/Fzo1, and the inner-membrane protein Opa1/Mgm1. In fungi, the outer-membrane protein Ugo1 functions in an adapter-like manner between Fzo1 and Mgm1. MIA: the mitochondrial intermembrane space import and assembly machinery (MIA) mediates the biogenesis of intermembrane space proteins that contain characteristic cysteine-rich internal signal sequences. This process involves the formation of transient intermolecular disulfides with the intermembrane space receptor Mia40 as well as the activity of the sulfhydryl oxidase Erv1. MINOS: the mitochondrial inner-membrane organizing system (MINOS) is involved in connecting the two subdomains of the inner mitochondrial membrane, the inner boundary membrane and the cristae. In yeast, MINOS consists of at least six different subunits: the mitofilin protein Fcj1, Mio10, Mio27, Aim5, Aim13 and Aim37. MINOS deficiency leads to detachment of cristae from the inner boundary membrane and loss of crista junctions. Additionally, MINOS components are engaged in multiple interactions with the outer mitochondrial membrane. Prohibitins: mitochondrial prohibitins belong to a large protein family also including flotillin and stomatin proteins, characterized by a so-called PHB domain. It has been suggested that these proteins are involved in the organization of membrane domains, most likely through specific lipid-binding properties. SAM: the sorting and assembly machinery (SAM complex) of the outer mitochondrial membrane mediates the membrane integration and folding of b-barrel proteins. SAM is composed of the membrane-integral Sam50 protein and the peripherally associated subunits Sam35 and Sam37. The morphology protein Mdm10 associates with SAM to support the assembly of some a-helical proteins. TOM: the translocase of the outer mitochondrial membrane (TOM complex) mediates the import of the vast majority of nuclear-encoded mitochondrial precursor proteins into the organelle. The protein-conducting channel of the TOM complex is formed by the b-barrel protein Tom40, which associates with several receptor proteins and small subunits involved in stabilization and dynamics of the TOM complex.
0962-8924/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2012.01.004 Trends in Cell Biology, April 2012, Vol. 22, No. 4
185
Opinion
Trends in Cell Biology April 2012, Vol. 22, No. 4
(a) Cytosol Outer membrane Intermembrane space
SAM
Porin
Ugo1
TOM
Mgm1
Mia40
Inner boundary membrane
MINOS
MINOS
TIM23
Crista junction
IV
Cristae membrane
H+
IV
H+
H+
III
H+
H+
III
+
II
Δψ
Mg m1
−
Matrix
III
H+
+
IV
H
III
H+ Fo
H+ Fo
F1
F1
(b)
Wild-type mitochondrion
(c)
MINOS
MitOS
Mitofilin/ Mitofilin/ Fcj1 Fcj1 Mio10
Mos1
MICOS
Yeast ORF name
Fcj1
YKR016W
Mcs10 YCL057C-A
MINOS mutant mitochondrion
MINOS Metazoa
Domain structure 1
+++
1
540
+++
Aim5
Aim5
Mcs12
YBR262C
1
Aim13
Aim13
Mcs19
YFR011C
1
MINOS1
Key:
97
Mitofilin/ IMMT/MINOS2
Signal sequence
106 Transmembrane domain
170
Coiled-coil region
Aim37
Aim37
Mcs27 YNL100W
1
234
Mitofilin domain
Mio27
Mos2
Mcs29
1
233
CHCH motif
CHCHD3/ MINOS3
MOMA-1 YGR235C
TRENDS in Cell Biology
Figure 1. Organization of the mitochondrial inner membrane. (a) Schematic view of mitochondrial outer and inner membranes. The inner membrane consists of two domains – the inner boundary membrane and the cristae membranes – which are connected by crista junctions. Proteins are differentially enriched in the two domains, although the distribution is not static but dynamic. Protein-transport machineries such as the presequence translocase (TIM23) and Mia40 are enriched in the inner boundary membrane, whereas respiratory complexes such as complexes II, III and IV and the F1Fo-ATP synthase are enriched in cristae membranes. Isoforms of the fusion protein Mgm1 are differentially enriched in inner boundary membrane and cristae membrane [97]. The mitochondrial inner-membrane organizing system (MINOS) is enriched in the vicinity of crista junctions. MINOS interacts with protein translocases of the outer membrane (TOM, SAM), the channel-forming protein porin (VDAC), the outer-membrane fusion component Ugo1, and the intermembrane space receptor Mia40. (b) The inner membrane of wild-type mitochondria forms invaginations (cristae). In MINOS mutant mitochondria, most crista junctions are lost and large internal membrane stacks, resembling a labyrinth, are observed. The ancient Greek drachma shows the head of Zeus or his son Minos and the mythical labyrinth of Knossos, Crete; in Greek mythology Minos was a king of Crete when the labyrinth was built. Photo of the drachma with permission from Fritz Rudolf Ku¨nker GmbH & Co. KG, Osnabru¨ck, www.kuenker.de, and Lu¨bke & Wiedemann, Stuttgart. (c) The MINOS complex contains six inner-membrane proteins. The nomenclatures in yeast (left) and metazoa (right) are indicated. ORF, open reading frame and systematic name in the yeast Saccharomyces cerevisiae. The domain structures of the MINOS subunits are shown; numbers refer to the first and last amino acid residues of the yeast proteins.
fusion (Mgm1/Opa1) (Figure 1a). The cristae membranes mainly contain the complexes of the respiratory chain, the F1Fo-ATP synthase, and metabolite carriers such as the ADP/ATP carrier [19,20]. Inner boundary membrane and cristae membranes are connected by tubular openings (Figure 1a,b). These 186
crista junctions (see Glossary) are believed to limit diffusion between both membrane domains, although there is no strict separation, but the distribution of molecules between the membrane domains is dynamic [17,20,22]. Crista junctions are also thought to limit diffusion of soluble molecules between the intracristal space and the remainder of the
Opinion intermembrane space, which is located between inner boundary membrane and outer membrane. Crista junctions thus help to form a microenvironment in the intracristal space that may enhance the capacity of the oxidative phosphorylation system of mitochondria [16,17,23–25]. Despite detailed morphological analyses of mitochondrial ultrastructure, little has been known about the molecules that are involved in the maintenance of cristae organization [16,17]. Recent studies by several groups led to the identification of a large protein complex that functions as a mitochondrial inner-membrane organizing system (MINOS) [26–29]. Surprisingly, MINOS interacts with a variety of different partner proteins, including several protein complexes of the mitochondrial outer membrane (Figure 1a). Here, we put the different observations into context and propose that MINOS forms a central core of a large organizing system that extends from ER–mitochondria junctions to mitochondrial outer and inner membranes and mitochondrial DNA (mtDNA) nucleoids. We term this organizing system ERMIONE, for ER–mitochondria organizing network, and suggest that ERMIONE functions as a regulatory system that includes both biochemical and genetic interactions. Mitochondrial inner-membrane organization Yeast mutants that affect the dimerization of the F1Fo-ATP synthase display altered cristae morphologies [23,29–33]. The F1Fo-ATP synthase can assemble into large spiral-like oligomeric structures that may form a scaffold for the stabilization of cristae structures, in particular for the formation of cristae tips [24,25,33,34]. However, the F1Fo-ATP synthase is probably not responsible for the formation or maintenance of crista junctions [33]. The inner-membrane protein Mgm1 (yeast)/Opa1 (mammals) is required for fusion of mitochondrial membranes [35–39]. In addition, mutants of Mgm1/Opa1 show altered cristae morphology. It is thought that oligomerization of Mgm1/Opa1 is required
Trends in Cell Biology April 2012, Vol. 22, No. 4
for normal cristae structure [37,39,40], although further work is needed to define the molecular mechanisms. The inner-membrane protein Mdm33 also affects innermembrane structure, possibly by regulating innermembrane division [41]. Characterization of the mitochondrial inner-membrane protein mitofilin was a major step in the analysis of crista junctions [26–29,33,42–44]. Mitofilin was initially identified in heart muscle cells [45] and received its name due to its colocalization with the filamentous mitochondrial network in fibroblasts [46]. Mitofilin was found in the proteome of purified mitochondria [47,48]. It is targeted to mitochondria by an N-terminal presequence and anchored in the inner membrane by a single hydrophobic transmembrane region that follows the presequence. The remainder of the protein is exposed to the mitochondrial intermembrane space and includes a coiled-coil region that supports protein–protein interactions (Figure 1c) [33,42]. Cells with decreased levels of mitofilin or mutant cells completely lacking mitofilin show a striking alteration of the inner morphology of mitochondria. The cristae membranes are detached from the inner boundary membrane and form large internal membrane stacks (Figure 1b) [33,42]. Because crista junctions are largely absent in mitofilin mutant cells, it was concluded that mitofilin is required for formation of crista junctions, and was therefore also termed Fcj1 in yeast [33]. However, crista junctions are not completely absent in mitofilin mutants [26,28] and thus mitofilin is not strictly essential for their formation. The available results are explained best by a role of mitofilin in the maintenance of crista junctions, including the prevention of cristae release from the inner boundary membrane. Though it was originally assumed that mitofilin mainly undergoes homotypic interactions [33,42], recent studies have led to the identification of a large hetero-oligomeric protein complex in the inner membrane that contains
Box 1. Identification of the mitochondrial inner-membrane organizing system Four independent studies identified a large protein complex of the inner mitochondrial membrane that is crucial for maintenance of the typical inner-membrane morphology. The complex consists of the core components mitofilin/Fcj1 and MINOS1/Mio10 together with at least four additional subunits. The complex has received three different names: MINOS, MICOS, and MitOS. All studies concordantly report that mutations affecting this protein complex induce a dramatic loss of crista junctions and a detachment of cristae membranes from the inner boundary membrane. von der Malsburg et al. [26] combined stable isotope-labeling with amino acids in cell culture (SILAC) with purification of mitofilincontaining protein complexes by affinity chromatography and mass spectrometric analysis. The identified protein complex is composed of mitofilin/Fcj1, Mio10, Mio27, Aim5, Aim13 and Aim37 and was termed mitochondrial inner-membrane organizing system (MINOS). This study also identified a physical interaction between mitofilin/ Fcj1 and the outer-membrane TOM complex. This interaction is important for protein import into the intermembrane space via the MIA pathway. Harner et al. [27] expressed a synthetic linker protein in yeast cells as a marker for contact sites between outer and inner mitochondrial membranes. They fractionated mitochondrial membranes and analyzed proteins enriched in contact-site fractions by mass spectrometry. Six of the proteins found in contact-site fractions were shown to form an inner-membrane complex preferentially
located at crista junctions, named the mitochondrial contact-site (MICOS) complex. This study provided evidence for interaction of this complex with the Ugo1 fusion component of the outer membrane and for an interaction with the SAM complex (in agreement with further studies [51–53]). Hoppins et al. [28] systematically analyzed genes involved in mitochondrial biogenesis and function for similar synthetic interaction patterns (MITO-MAP). One gene cluster including FCJ1 encoded subunits of a protein complex required for maintenance of mitochondrial inner-membrane morphology, termed mitochondrial organizing structure (MitOS). MitOS genes showed strong genetic interactions with ERMES and phospholipid biosynthesis genes. Fluorescence microscopy analysis of cells expressing GFP-tagged subunits suggested the formation of large structures spanning the mitochondrial intermembrane space. Alkhaja et al. [29] identified Mio10 as a protein potentially stabilizing mitochondrial membrane protein supercomplexes. Tagged Mio10 co-purified mitofilin/Fcj1 and further components of MINOS. The human ortholog of Mio10 is also part of a mitofilincontaining complex and was termed MINOS1. Further components are the CHCHD3/MINOS3 protein, which belongs to the same protein family as yeast Aim13, as well as HSPA9 and the J-protein DnaJC11. In agreement with further studies [51–53], this work demonstrated a direct interaction of the MINOS complex with the SAM complex of the outer mitochondrial membrane. 187
Opinion
Trends in Cell Biology April 2012, Vol. 22, No. 4
mitofilin as one of the core components (Figure 1a,c; Box 1) [26–29]. This large mitofilin complex was termed mitochondrial inner-membrane organizing system (MINOS) [26,29]. It is a remarkable coincidence that MINOS was identified in parallel by four independent studies. Thus the complex received the further names MICOS (mitochondrial contact site) and MitOS (mitochondrial organizing structure) [27,28]. Different experimental strategies and approaches led to the identification of MINOS/MICOS/MitOS (Box 1) [26–29]. The MINOS complex contains six different subunits (Figures 1c, 2). Mitofilin/Fcj1 and the small integral inner-membrane protein Mio10 are the two core components [26–29]. The absence of either leads to dissociation of the MINOS complex and massive rearrangement of inner-membrane morphology, with the loss of crista junctions and the appearance of large internal membrane stacks. Three MINOS subunits were originally identified in a genetic screen for proteins that are involved in the inheritance of mtDNA,
and were termed Aim5, Aim13 and Aim37 (altered inheritance of mitochondria) (currently, more than 40 AIM genes are listed in the Saccharomyces genome database, although the molecular function of many of them is not yet known) [49]. The three Aim proteins of the MINOS complex are all inner-membrane proteins, as are mitofilin and Mio10. Aim5 and Aim37 are integral membrane proteins, whereas Aim13 is a peripheral membrane protein (Figure 1c). Mutants of these AIM genes show phenotypes that are in principle similar to those of mutants lacking mitofilin or Mio10; however, the defects are not as severe. Lack of Aim proteins leads to a partial dissociation of the MINOS complex, a reduction of the number of crista junctions, and a partial alteration of mitochondrial morphology (with internal cristae stacks) [26–28]. The sixth subunit of the MINOS complex, Mio27, is related to Aim37; Mio27 mutants show only mild phenotypes. The recently identified C. elegans mitochondrial morphology protein MOMA-1 is an ortholog of Aim37 and Mio27 [44] (Figure 1c).
Endoplasmic reticulum
Cytosol
Mitochondrion
ER lumen
ER membrane Cytosol OM IMS IM
Gem Mmm 1 12 1
Lipid transport
34 Mdm 10
Prohibitins
Mfn 2 Mfn 1/2
ERMES Tom7 Mdm 10
Mdm 10 SAM
13 Fcj1 5Aim 37 10 27 Mio
Mdm 31 32
MINOS complexes
Matrix
Porin
Crista junction
Ca++ flux
TOM Opa1/ Mgm1
Mia40 TIM23
MINOS complexes
Nucleoid TRENDS in Cell Biology
Figure 2. An ER–mitochondria organizing network (ERMIONE). The ER and the mitochondrial outer membrane, as well as the mitochondrial outer and inner membranes, are connected by protein–protein interactions. The lower part of the figure shows a schematic view of protein complexes that are involved in membrane contact sites. Biochemically demonstrated interactions are indicated by double arrows; genetic interactions are indicated by dotted double arrows. ER and mitochondria can be physically connected by the ERMES complex and by mitofusins (Mfn). MINOS complexes probably form an extended network on the intermembrane space side of the inner membrane. MINOS interacts with several outer-membrane proteins, including the protein translocases TOM and SAM, the abundant channel-protein porin, and fusion components. MINOS transiently interacts with Mia40 and promotes protein import through TOM into the intermembrane space. MINOS and ERMES have been linked genetically by analysis of double mutants and biochemically via the SAM complex (SAM interacts with MINOS, and the morphology protein Mdm10 is present in both SAM and ERMES). ERMES, MINOS and further inner-membrane proteins, including prohibitins and Mdm31/32, are involved in the maintenance of mitochondrial DNA that is organized in nucleoids. The network of ER–mitochondrial outer-membrane–inner-membrane interactions is termed the ER–mitochondria organizing network (ERMIONE). This network of interactions promotes the transport of lipids and calcium ions between the compartments. OM, outer membrane; IMS, intermembrane space; IM, inner membrane.
188
Opinion The MINOS complex forms a large structure in the megadalton range and thus its subunits are probably present in multiple copies (Figure 2). Mitofilin and Mio10 contain sequence elements that favor protein– protein interactions and probably form homo- and hetero-oligomeric structures. Affinity-purification studies yielded various combinations of MINOS components, suggesting that multiple subcomplexes may exist that are arranged in a large, dynamic superstructure [26–29]. MINOS components are enriched in the vicinity of crista junctions [27,33], and all subunits contain domains that are exposed to the intermembrane space. Taking the biochemical and morphological evidence together [26–29], MINOS may form an extended network on the intermembrane space side of the inner membrane that is important for the maintenance of inner-membrane organization. The formation of the typical cristae structures probably involves several dynamic and structural elements, including MINOS, the F1Fo-ATP synthase, and Mgm1/Opa1. Multiple interactions of MINOS with the mitochondrial outer membrane Interactions of the MINOS complex are not limited to the mitochondrial inner membrane. Several studies surprisingly demonstrated that proteins and protein complexes of the mitochondrial outer membrane physically interact with MINOS components. The outer membrane contains two major machineries for protein transport – the TOM complex that is responsible for importing hundreds of different nuclear-encoded proteins into mitochondria, and the SAM complex (TOB complex) that mediates the insertion of b-barrel proteins into the outer membrane [1,8,9,50]. Both translocase machineries were found as interaction partners of MINOS (Figures 1a,2) [26–29, 51–53]. In the case of the TOM complex, MINOS indeed plays a supportive role in protein import. However, MINOS does not promote the import of precursor proteins into or across the inner membrane; the presequence translocase (TIM23 complex) and the carrier translocase (TIM22 complex) of the inner membrane have not been found in association with MINOS. Instead, MINOS transiently interacts with the intermembrane space import receptor Mia40 [26]. Mia40 is the core of the mitochondrial intermembrane-space assembly machinery that drives the import of proteins into the intermembrane space by an oxidative mechanism [9,54–57]. Mia40 directly interacts with the incoming substrate proteins via disulfide bonds. To capture immediately preproteins in transit through the TOM channel, Mia40 binds to the signal sequences of incoming preproteins as soon as they emerge on the intermembrane space side of the TOM complex [26]. For this task, Mia40 has to be positioned in close vicinity to the TOM complex, and this is achieved by the transient interaction of mitofilin with both the TOM complex and Mia40. Mitofilin may thus perform adapter-like functions to link different machineries of the mitochondrial protein-import apparatus. The exact molecular function of the interaction of MINOS with the SAM complex has not yet been defined. An adapter/organizing role of MINOS may support the cooperation of TOM and SAM, and thus MINOS may assist in the transfer of b-barrel proteins from the TOM complex
Trends in Cell Biology April 2012, Vol. 22, No. 4
to the SAM complex. In cells with reduced levels of Sam50, the cristae architecture was altered [53], suggesting that the interaction of MINOS with the outer membrane may be important for maintaining inner-membrane architecture. In addition to the association with protein translocases, MINOS was also found to interact with the most abundant outer-membrane protein porin (VDAC), as well as with the fusion component Ugo1 (Figures 1a, 2) [27,28]. Porin is the major channel for transport of metabolites between the cytosol and the intermembrane space. Because metabolites are differentially distributed between intracristal space and the remainder of the intermembrane space [14,16, 58], the enrichment of MINOS in the vicinity of crista junctions may help to position the main metabolite channel of mitochondria close to crista junctions, and may thus facilitate the efficient distribution of metabolites. Ugo1 is a subunit of the outer-membrane fusion machinery of yeast mitochondria and has an adapter-like function through its linkage of two dynamin-like GTPases: the outermembrane protein Fzo1 (termed mitofusin in higher eukaryotes) and Mgm1/Opa1 of the inner membrane [59–61]. Although it is an open question whether MINOS is present in a supercomplex with Fzo1 and Mgm1, or if MINOS interacts with Ugo1 only [26,27,52], the interaction of MINOS with Ugo1 provides a link between two machineries that control mitochondrial membrane morphology and dynamics. An ER–mitochondria organizing network The large number of reported MINOS interaction partners of seemingly unrelated functions raises the question of how these findings can be put into a functional context. We propose that MINOS forms a central core of a large organizing network of mitochondria that includes numerous further components. The large size of MINOS may provide a scaffold for multiple binding partners and thus help to spatially organize and coordinate mitochondrial functions (Figure 2). Of particular importance is the functional connection of SAM and TOM to the ER–mitochondria encounter structure (ERMES) [62–65]. In fungi, ERMES physically connects ER and mitochondria by containing both an ER-located subunit (Mmm1) and mitochondrial-located subunits within the same complex (Figure 2). Remarkably, the morphology protein Mdm10 of the mitochondrial outer membrane has dual localization in SAM and ERMES [66–70]. A small TOM subunit also exhibits dual localization: Tom7 is present in the TOM complex, and also associates with Mdm10, and thus regulates the distribution of Mdm10 between SAM and ERMES [67,69,71]. Yeast mutants of ERMES components show a dramatic alteration of mitochondrial shape, leading to giant ball-like mitochondrial structures [67,68,72–79]. Yeast mutants of SAM components as well as of several TOM components lead to a similar phenotype [66,67], underscoring the functional connection between ERMES, SAM and TOM. Remarkably, mutants of the ER-located subunit Mmm1 were also reported to affect the ultrastructure of the mitochondrial inner membrane, leading to a massive disorganization of cristae membranes [80], suggesting an important connection from the ER to the mitochondrial 189
Opinion inner membrane. Indeed, strong genetic interactions have been observed between ERMES and MINOS components [28]. Taken together, we propose that ERMES, SAM, TOM and MINOS are connected in a dynamic network and thus link ER to mitochondrial outer and inner membranes. ERMES as well as MINOS have been implicated in lipid transfer and metabolism [2,27,28,62–65,81] and thus they may provide a scaffold for transport of lipid molecules between ER and both mitochondrial membranes [82]. Both complexes are genetically linked to the prohibitin ring complexes of the inner membrane that are integrated into a network of mitochondrial lipid metabolism [2,28,81]. In addition, prohibitins affect cristae morphogenesis by regulating the processing of the fusion protein Mgm1/ Opa1 [83]. The close connections between ER and mitochondria probably facilitate the uptake of calcium ions by mitochondria upon their release from the ER [84]. In higher eukaryotes, mitofusins have been reported to link physically ER and mitochondria and thus to promote calcium transfer into mitochondria (Figure 2) [85]. It is an open question whether ERMES and mitofusin tethers exist in parallel in the same organism. Interestingly, in yeast MINOS interacts with the fusion component Ugo1 that binds to Fzo1, the yeast ortholog of mitofusins [27,59]. Although an ortholog of Ugo1 has not yet been found in higher eukaryotes, it is tempting to speculate that MINOS, whose core components are conserved from yeast to human (Figure 1c), is the connecting partner of both systems that link mitochondria and the ER, providing a communication chain across three organellar membranes. ERMES has also been proposed to be involved in mitochondrial distribution and the connection of mitochondria to the cytoskeleton [73–75], although different views exist on the molecular mechanism. The recent identification of the GTPase Gem1 as a mitochondria-bound subunit of ERMES [86,87] provides an interesting twist because the protein Miro (mitochondrial Rho-like), the homolog of Gem1 in higher eukaryotes, functions as an adapter in linking mitochondria to the cytoskeleton [88–90]. Four of the MINOS subunits were found in a genetic screen for altered inheritance of mitochondria, reflected in the names Aim5, Aim13 and Aim37; the fourth, mitofilin/ Fcj1, was also termed Aim28 [49]. MINOS is thus involved in the maintenance of mtDNA. Yeast mtDNA is organized into DNA–protein complexes termed nucleoids, and ERMES can be found in foci that are adjacent to a subset of nucleoids [75,78,80,91]. Genetic interaction studies revealed a functional link between ERMES, MINOS and further inner-membrane proteins that are involved in regulating mitochondrial morphology and the maintenance of mtDNA. These include the morphology proteins Mdm31 and Mdm32 that are anchored in the inner membrane and expose large domains to the intermembrane space [79]. Yeast mutants of Mdm31 and Mdm32 show a similar alteration of mitochondrial shape as ERMES, SAM and TOM mutants (i.e. large spherical mitochondria), and Mdm31 and Mdm32 are required to organize mtDNA into nucleoids [79]. It is thus likely that MINOS, ERMES and further Mdm proteins functionally cooperate in the maintenance and organization of mtDNA (Figure 2). 190
Trends in Cell Biology April 2012, Vol. 22, No. 4
Box 2. Outstanding questions What is the mechanistic role of MINOS in the formation and maintenance of crista junctions? What are the physiological consequences of the segregation of cristae tubules from the inner boundary membrane in MINOS mutants? How dynamic are the ERMES and MINOS complexes and which factors regulate their assembly state? Do ERMES and MINOS directly interact with each other or are further components involved? How are the different phenotypes associated with mutations of ERMIONE components, including membrane morphology, lipid metabolism, protein biogenesis and mtDNA maintenance, related to each other at the molecular level? Are subcomplexes of MINOS or ERMES responsible for distinct functions? Are MINOS or ERMES involved in mitochondrial or ER functions during programmed cell death? Is there a signaling pathway extending from the mitochondrial matrix via MINOS and ERMES to the ER lumen and eventually to the nucleus?
Taken together, we propose that ERMES and MINOS are linked into an ER–mitochondria organizing network (ERMIONE) that includes machineries for protein biogenesis, lipid metabolism, metabolite and ion transport, and mtDNA organization. ER–mitochondria interaction sites have also been shown to be related to sites of mitochondrial division [92] although the molecular mechanisms are not yet known. Cristae remodeling can occur during mitochondrial steps of apoptosis [16,40,93–96]. Future studies will need to address whether MINOS, ERMES and further components of ERMIONE play a role in mitochondrial fission and apoptotic events. Concluding remarks Morphological and genetic studies led to the suggestion that there could be a multi-membrane-spanning connection from ERMES across the mitochondrial membranes to mtDNA nucleoids [63,64,75,76,79,80,91]. However, only limited information was available on the molecular composition and structural arrangement of such an organizing center. The identification of MINOS and its multiple interactions provide a new view on mitochondrial architecture and biogenesis. MINOS forms a large scaffold and interaction platform that connects to outer-membrane complexes, including ERMES, as well as to machineries inside mitochondria. The hypothesis of a large ER–mitochondria organizing network (ERMIONE) will provide a platform for research on the integrative organization of mitochondrial functions and a number of exciting questions for future research remain (Box 2). Acknowledgments This work has been supported by the Deutsche Forschungsgemeinschaft, the Excellence Initiative of the German Federal & State Governments (EXC 294), the Gottfried Wilhelm Leibniz Program, Sonderforschungsbereich 746, Landesforschungspreis Baden-Wu¨rttemberg, and the Bundesministerium fu¨r Bildung und Forschung.
References 1 Dolezal, P. et al. (2006) Evolution of the molecular machines for protein import into mitochondria. Science 313, 314–318 2 Osman, C. et al. (2011) Making heads or tails of phospholipids in mitochondria. J. Cell Biol. 192, 7–16
Opinion 3 Otera, H. and Mihara, K. (2011) Molecular mechanisms and physiologic functions of mitochondrial dynamics. J. Biochem. 149, 241–251 4 Scheffler, I.E. (2008) Mitochondria (2nd edn), J. Wiley and Sons 5 McBride, H.M. et al. (2006) Mitochondria: more than just a powerhouse. Curr. Biol. 16, R551–R560 6 Wang, C. and Youle, R.J. (2009) The role of mitochondria in apoptosis. Annu. Rev. Genet. 43, 95–118 7 Lill, R. (2009) Function and biogenesis of iron–sulphur proteins. Nature 460, 831–838 8 Neupert, W. and Herrmann, J.M. (2007) Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 9 Chacinska, A. et al. (2009) Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644 10 Okamoto, K. and Shaw, J.M. (2005) Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu. Rev. Genet. 39, 503–536 11 Westermann, B. (2010) Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell Biol. 11, 872–884 12 Palmer, C.S. et al. (2011) The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery. Cell Signal. 23, 1534–1545 13 Perkins, G. et al. (1997) Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts. J. Struct. Biol. 119, 260–272 14 Frey, T.G. and Mannella, C.A. (2000) The internal structure of mitochondria. Trends Biochem. Sci. 25, 319–324 15 Frey, T.G. et al. (2002) Insight into mitochondrial structure and function from electron tomography. Biochim. Biophys. Acta 1555, 196–203 16 Mannella, C.A. (2006) The relevance of mitochondrial membrane topology to mitochondrial function. Biochim. Biophys. Acta 1762, 140–147 17 Zick, M. et al. (2009) Cristae formation – linking ultrastructure and function of mitochondria. Biochim. Biophys. Acta 1793, 5–19 18 Werner, S. and Neupert, W. (1972) Functional and biogenetical heterogeneity of the inner membrane of rat-liver mitochondria. Eur. J. Biochem. 25, 379–396 19 Gilkerson, R.W. et al. (2003) The cristal membrane of mitochondria is the principal site of oxidative phosphorylation. FEBS Lett. 546, 355–358 20 Vogel, F. et al. (2006) Dynamic subcompartmentalization of the mitochondrial inner membrane. J. Cell Biol. 175, 237–247 21 Wurm, C.A. and Jakobs, S. (2006) Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast. FEBS Lett. 580, 5628–5634 22 Suppanz, I.E. et al. (2009) The m-AAA protease processes cytochrome c peroxidase preferentially at the inner boundary membrane of mitochondria. Mol. Biol. Cell 20, 572–580 23 Bornho¨vd, C. et al. (2006) Mitochondrial membrane potential is dependent on the oligomeric state of F1Fo-ATP synthase supracomplexes. J. Biol. Chem. 281, 13990–13998 24 Strauss, M. et al. (2008) Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J. 27, 1154–1160 25 Davies, K.M. et al. (2011) Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc. Natl. Acad. Sci. U.S.A. 108, 14121–14126 26 von der Malsburg, K. et al. (2011) Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis. Dev. Cell 21, 694–707 27 Harner, M. et al. (2011) The mitochondrial contact site complex, a determinant of mitochondrial architecture. EMBO J. 30, 4356–4370 28 Hoppins, S. et al. (2011) A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria. J. Cell Biol. 195, 323–340 29 Alkhaja, A.K. et al. (2012) MINOS1 is a conserved component of mitofilin complexes and required for mitochondrial function and cristae organization. Mol. Biol. Cell 23, 247–257 30 Giraud, M.F. et al. (2002) Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? Biochim. Biophys. Acta 1555, 174–180 31 Paumard, P. et al. (2002) The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J. 21, 221–230 32 Arselin, G. et al. (2004) The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology. J. Biol. Chem. 279, 40392–40399
Trends in Cell Biology April 2012, Vol. 22, No. 4
33 Rabl, R. et al. (2009) Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g. J. Cell Biol. 185, 1047–1063 34 Buzhynskyy, N. et al. (2007) Rows of ATP synthase dimers in native mitochondrial inner membranes. Biophys. J. 93, 2870–2876 35 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 36 Cipolat, S. et al. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U.S.A. 101, 15927–15932 37 Meeusen, S. et al. (2006) Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Cell 127, 383–395 38 Ishihara, N. et al. (2006) Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. EMBO J. 25, 2966–2977 39 DeVay, R.M. et al. (2009) Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion. J. Cell Biol. 186, 793–803 40 Frezza, C. et al. (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126, 177–189 41 Messerschmitt, M. et al. (2003) The inner membrane protein Mdm33 controls mitochondrial morphology in yeast. J. Cell Biol. 160, 553–564 42 John, G.B. et al. (2005) The mitochondrial inner membrane protein mitofilin controls cristae morphology. Mol. Biol. Cell 16, 1543–1554 43 Mun, J.Y. et al. (2010) Caenorhabditis elegans mitofilin homologs control the morphology of mitochondrial cristae and influence reproduction and physiology. J. Cell. Physiol. 224, 748–756 44 Head, B.P. et al. (2011) A novel mitochondrial outer membrane protein, MOMA-1, that affects cristae morphology in Caenorhabditis elegans. Mol. Biol. Cell 22, 831–841 45 Icho, T. et al. (1994) A novel human gene that is preferentially transcribed in heart muscle. Gene 144, 301–306 46 Odgren, P.R. et al. (1996) Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains. J. Cell Sci. 109, 2253–2264 47 Sickmann, A. et al. (2003) The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U.S.A. 100, 13207–13212 48 Taylor, S.W. et al. (2003) Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 21, 281–286 49 Hess, D.C. et al. (2009) Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis. PLoS Genet. 5, e1000407 50 Dukanovic, J. and Rapaport, D. (2011) Multiple pathways in the integration of proteins into the mitochondrial outer membrane. Biochim. Biophys. Acta 1808, 971–980 51 Xie, J. et al. (2007) The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiledcoil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11. FEBS Lett. 581, 3545–3549 52 Darshi, M. et al. (2011) ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function. J. Biol. Chem. 286, 2918–2932 53 Ott, C. et al. (2012) Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes. Mol. Cell. Biol. 32, 1173–1188 54 Koehler, C.M. and Tienson, H.L. (2009) Redox regulation of protein folding in the mitochondrial intermembrane space. Biochim. Biophys. Acta 1793, 139–145 55 Herrmann, J.M. and Riemer, J. (2012) The mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space. J. Biol. Chem. 287, 4426–4433 56 Sideris, D.P. and Tokatlidis, K. (2010) Oxidative protein folding in the mitochondrial intermembrane space. Antioxid. Redox Signal. 13, 1189–1204 57 Endo, T. et al. (2010) Structural basis for the disulfide relay system in the mitochondrial intermembrane space. Antioxid. Redox Signal. 13, 1359–1373 58 Sukhorukov, V.M. and Bereiter-Hahn, J. (2009) Anomalous diffusion induced by cristae geometry in the inner mitochondrial membrane. PLoS ONE 4, e4604 59 Sesaki, H. and Jensen, R.E. (2004) Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion. J. Biol. Chem. 279, 28298–28303 191
Opinion 60 Hoppins, S. et al. (2009) Mitochondrial outer and inner membrane fusion requires a modified carrier protein. J. Cell Biol. 184, 569–581 61 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 62 Kornmann, B. et al. (2009) An ER–mitochondrial tethering complex revealed by a synthetic biology screen. Science 325, 477–481 63 Wiedemann, N. et al. (2009) Cell biology: connecting organelles. Science 325, 403–404 64 Kornmann, B. and Walter, P. (2010) ERMES-mediated ER– mitochondria contacts: molecular hubs for the regulation of mitochondrial biology. J. Cell Sci. 123, 1389–1393 65 Elbaz, Y. and Schuldiner, M. (2011) Staying in touch: the molecular era of organelle contact sites. Trends Biochem. Sci. 36, 616–623 66 Meisinger, C. et al. (2004) The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane. Dev. Cell 7, 61–71 67 Meisinger, C. et al. (2006) Mitochondrial protein sorting: differentiation of b-barrel assembly by Tom7-mediated segregation of Mdm10. J. Biol. Chem. 281, 22819–22826 68 Meisinger, C. et al. (2007) The morphology proteins Mdm12/Mmm1 function in the major b-barrel assembly pathway of mitochondria. EMBO J. 26, 2229–2239 69 Yamano, K. et al. (2010) Tom7 regulates Mdm10-mediated assembly of the mitochondrial import channel protein Tom40. J. Biol. Chem. 285, 41222–41231 70 Wideman, J.G. et al. (2010) Roles of the Mdm10, Tom7, Mdm12, and Mmm1 proteins in the assembly of mitochondrial outer membrane proteins in Neurospora crassa. Mol. Biol. Cell 21, 1725–1736 71 Becker, T. et al. (2011) Biogenesis of mitochondria: dual role of Tom7 in modulating assembly of the preprotein translocase of the outer membrane. J. Mol. Biol. 405, 113–124 72 Burgess, S.M. et al. (1994) MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria. J. Cell Biol. 126, 1375–1391 73 Sogo, L.F. and Yaffe, M.P. (1994) Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane. J. Cell Biol. 126, 1361–1373 74 Berger, K.H. et al. (1997) Mdm12p, a component required for mitochondrial inheritance that is conserved between budding and fission yeast. J. Cell Biol. 136, 545–553 75 Boldogh, I.R. et al. (2003) A protein complex containing Mdm10p, Mdm12p, and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery. Mol. Biol. Cell 14, 4618–4627 76 Kondo-Okamoto, N. et al. (2003) Mmm1p spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation. J. Biol. Chem. 278, 48997–49005 77 Frederick, R.L. et al. (2004) Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway. J. Cell Biol. 167, 87–98 78 Youngman, M.J. et al. (2004) Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids. J. Cell Biol. 164, 677–688
192
Trends in Cell Biology April 2012, Vol. 22, No. 4
79 Dimmer, K.S. et al. (2005) Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast. J. Cell Biol. 168, 103–115 80 Hobbs, A.E. et al. (2001) Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability. J. Cell Biol. 152, 401–410 81 Osman, C. et al. (2009) The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria. J. Cell Biol. 184, 583–596 82 Achleitner, G. et al. (1999) Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur. J. Biochem. 264, 545–553 83 Merkwirth, C. et al. (2008) Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria. Genes Dev. 22, 476–488 84 Szabadkai, G. et al. (2006) Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J. Cell Biol. 175, 901–911 85 de Brito, O.M. and Scorrano, L. (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456, 605–610 86 Kornmann, B. et al. (2011) The conserved GTPase Gem1 regulates endoplasmic reticulum–mitochondria connections. Proc. Natl. Acad. Sci. U.S.A. 108, 14151–14156 87 Stroud, D.A. et al. (2011) Composition and topology of the endoplasmic reticulum-mitochondria encounter structure. J. Mol. Biol. 413, 743–750 88 Glater, E.E. et al. (2006) Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent. J. Cell Biol. 173, 545–557 89 Wang, X. and Schwarz, T.L. (2009) The mechanism of Ca2+-dependent regulation of kinesin-mediated mitochondrial motility. Cell 136, 163–174 90 Koshiba, T. et al. (2011) Structure–function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance. J. Biol. Chem. 286, 354–362 91 Meeusen, S. and Nunnari, J. (2003) Evidence for a two membranespanning autonomous mitochondrial DNA replisome. J. Cell Biol. 163, 503–510 92 Friedman, J.R. et al. (2011) ER tubules mark sites of mitochondrial division. Science 334, 358–362 93 Scorrano, L. et al. (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell 2, 55–67 94 Karbowski, M. and Youle, R.J. (2003) Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ. 10, 870–880 95 Germain, M. et al. (2005) Endoplasmic reticulum BIK initiates DRP1regulated remodelling of mitochondrial cristae during apoptosis. EMBO J. 24, 1546–1556 96 Yamaguchi, R. et al. (2008) Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization. Mol. Cell 31, 557–569 97 Zick, M. et al. (2009) Distinct roles of the two isoforms of the dynamin-like GTPase Mgm1 in mitochondrial fusion. FEBS Lett. 583, 2237–2243