Role of MINOS in Mitochondrial Membrane Architecture: Cristae Morphology and Outer Membrane Interactions Differentially Depend on Mitofilin Domains

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.     

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component?of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.     

ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.     

A Highlights from MBoC Selection: Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.     

Mitofilin对线粒体功能和疾病发生的影响

Mitofilin是一种线粒体内膜蛋白,与多种线粒体蛋白相互作用,共同参与线粒体内膜嵴形态的维持、线粒体内蛋白质的转运过程等。干扰mitofilin~表达不仅引起线粒体结构的异常,而且明显抑制了线粒体功能的正常发挥。最近的研究表明,在多种疾病中mitonlin都异常表达,从而导致线粒体结构的完整性和功能的障碍,促进了疾病的发生发展。     

CHCM1/CHCHD6, novel mitochondrial protein linked to regulation of mitofilin and mitochondrial cristae morphology

The structural integrity of mitochondrial cristae is crucial for mitochondrial functions; however, the molecular events controlling the structural integrity and biogenesis of mitochondrial cristae remain to be fully elucidated. Here, we report the functional characterization of a novel mitochondrial protein named CHCM1 (coiled coil helix cristae morphology 1)/CHCHD6. CHCM1/CHCHD6 harbors a coiled coil helix-coiled coil helix domain at its C-terminal end and predominantly localizes to mitochondrial inner membrane. CHCM1/CHCHD6 knockdown causes severe defects in mitochondrial cristae morphology. The mitochondrial cristae in CHCM1/CHCHD6-deficient cells become hollow with loss of structural definitions and reduction in electron-dense matrix. CHCM1/CHCHD6 depletion also leads to reductions in cell growth, ATP production, and oxygen consumption. CHCM1/CHCHD6 through its C-terminal end strongly and directly interacts with the mitochondrial inner membrane protein mitofilin, which is known to also control mitochondrial cristae morphology. CHCM1/CHCHD6 also interacts with other mitofilin-associated proteins, including DISC1 and CHCHD3. Knockdown of CHCM1/CHCHD6 reduces mitofilin protein levels; conversely, mitofilin knockdown leads to reduction in CHCM1 levels, suggesting coordinate regulation between these proteins. Our results further indicate that genotoxic anticancer drugs that induce DNA damage down-regulate CHCM1/CHCHD6 expression in multiple human cancer cells, whereas mitochondrial respiratory chain inhibitors do not affect CHCM1/CHCHD6 levels. CHCM1/CHCHD6 knockdown in human cancer cells enhances chemosensitivity to genotoxic anticancer drugs, whereas its overexpression increases resistance. Collectively, our results indicate that CHCM1/CHCHD6 is linked to regulation of mitochondrial cristae morphology, cell growth, ATP production, and oxygen consumption and highlight its potential as a possible target for cancer therapeutics.     

The C-terminal domain of Fcj1 is required for formation of crista junctions and interacts with the TOB/SAM complex in mitochondria

Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of β-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex.     

The inner membrane protein Mdm33 controls mitochondrial morphology in yeast

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.     

C1orf163/RESA1 Is a Novel Mitochondrial Intermembrane Space Protein Connected to Respiratory Chain Assembly

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60 kDa and 150 kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

A novel mitochondrial outer membrane protein, MOMA-1, that affects cristae morphology in Caenorhabditis elegans

Three proteins with similar effects on mitochondrial morphology were identified in an RNA interference (RNAi) screen for mitochondrial abnormalities in Caenorhabditis elegans. One of these is the novel mitochondrial outer membrane protein MOMA-1. The second is the CHCHD3 homologue, CHCH-3, a small intermembrane space protein that may act as a chaperone. The third is a mitofilin homologue, IMMT-1. Mitofilins are inner membrane proteins that control the shapes of cristae. RNAi or mutations in each of these genes change the relatively constant diameters of mitochondria into highly variable diameters, ranging from thin tubes to localized swellings. Neither growth nor brood size of the moma-1, chch-3, or immt-1 single mutants is affected, suggesting that their metabolic functions are normal. However, growth of moma-1 or immt-1 mutants on chch-3(RNAi) leads to withered gonads, a lack of mitochondrial staining, and a dramatic reduction in fecundity, while moma-1; immt-1 double mutants are indistinguishable from single mutants. Mutations in moma-1 and immt-1 also have similar effects on cristae morphology. We conclude that MOMA-1 and IMMT-1 act in the same pathway. It is likely that the observed effects on mitochondrial diameter are an indirect effect of disrupting cristae morphology.     

Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes

Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of β-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).     

New insights into mitochondrial fusion

Fusion controls mitochondrial morphology and is important for normal mitochondrial function, including roles in respiration, development, and apoptosis. Key components of the mitochondrial fusion machinery have been identified, allowing an initial dissection of its molecular mechanism. Outer and inner membrane fusion events are coordinately coupled but are mechanistically distinct. Mitofusins are mitochondrial GTPases that likely mediate outer membrane fusion. The dynamin-related protein OPA1/Mgm1p is required for inner membrane fusion and maintenance of normal cristae structure. We highlight recent findings that have advanced our understanding of the mechanism, function, and regulation of mitochondrial fusion.     

Detailed Analysis of the Human Mitochondrial Contact Site Complex Indicate a Hierarchy of Subunits

Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.     

A Splicing Mutation in the Novel Mitochondrial Protein DNAJC11 Causes Motor Neuron Pathology Associated with Cristae Disorganization,and Lymphoid Abnormalities in Mice

Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.     

The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11

A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.     

The relevance of mitochondrial membrane topology to mitochondrial function

This review summarizes recent findings from electron tomography about the three-dimensional shape of mitochondrial membranes and its possible influence on a range of mitochondrial functions. The inner membrane invaginations called cristae are pleomorphic, typically connected by narrow tubular junctions of variable length to the inner boundary membrane. This design may restrict intra-mitochondrial diffusion of metabolites such as ADP, and of soluble proteins such as cytochrome c. Tomographic images of a variety of mitochondria suggest that inner membrane topology reflects a balance between membrane fusion and fission. Proteins that can affect cristae morphology include tBid, which triggers cytochrome c release in apoptosis, and the dynamin-like protein Mgm1, involved in inter-mitochondrial membrane fusion. In frozen-hydrated rat-liver mitochondria, the space between the inner and outer membranes contains 10-15 nm particles that may represent macromolecular complexes involved in activities that span the two membranes.     

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

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

Effects of lipids on mitochondrial functions

Mitochondria contain two membranes: the outer and inner membrane. Whereas the outer membrane is particularly enriched in phospholipids, the inner membrane has an unusual high protein content and forms large invaginations termed cristae. The proper phospholipid composition of the membranes is crucial for mitochondrial functions. Phospholipids affect activity, biogenesis and stability of protein complexes including protein translocases and respiratory chain supercomplexes. Negatively charged phospholipids such as cardiolipin are important for the architecture of the membranes and recruit soluble factors to the membranes to support mitochondrial dynamics. Thus, phospholipids not only form the hydrophobic core of biological membranes that surround mitochondria, but also create a specific environment to promote functions of various protein machineries. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.