Sam50-Mic19-Mic60 axis determines mitochondrial cristae architecture by mediating mitochondrial outer and inner membrane contact.
ABSTRACT: Mitochondrial cristae are critical for efficient oxidative phosphorylation, however, how cristae architecture is precisely organized remains largely unknown. Here, we discovered that Mic19, a core component of MICOS (mitochondrial contact site and cristae organizing system) complex, can be cleaved at N-terminal by mitochondrial protease OMA1 under certain physiological stresses. Mic19 directly interacts with mitochondrial outer-membrane protein Sam50 (the key subunit of SAM complex) and inner-membrane protein Mic60 (the key component of MICOS complex) to form Sam50-Mic19-Mic60 axis, which dominantly connects SAM and MICOS complexes to assemble MIB (mitochondrial intermembrane space bridging) supercomplex for mediating mitochondrial outer- and inner-membrane contact. OMA1-mediated Mic19 cleavage causes Sam50-Mic19-Mic60 axis disruption, which separates SAM and MICOS and leads to MIB disassembly. Disrupted Sam50-Mic19-Mic60 axis, even in the presence of SAM and MICOS complexes, causes the abnormal mitochondrial morphology, loss of mitochondrial cristae junctions, abnormal cristae distribution and reduced ATP production. Importantly, Sam50 displays punctate distribution at mitochondrial outer membrane, and acts as an anchoring point to guide the formation of mitochondrial cristae junctions. Therefore, we propose that Sam50-Mic19-Mic60 axis-mediated SAM-MICOS complexes integration determines mitochondrial cristae architecture.
Project description: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.
Project description:Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome <i>c</i> oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs.
Project description:The MICOS complex (mitochondrial contact site and cristae organizing system) is essential for mitochondrial inner membrane organization and mitochondrial membrane contacts, however, the molecular regulation of MICOS assembly and the physiological functions of MICOS in mammals remain obscure. Here, we report that Mic60/Mitofilin has a critical role in the MICOS assembly, which determines the mitochondrial morphology and mitochondrial DNA (mtDNA) organization. The downregulation of Mic60/Mitofilin or Mic19/CHCHD3 results in instability of other MICOS components, disassembly of MICOS complex and disorganized mitochondrial cristae. We show that there exists direct interaction between Mic60/Mitofilin and Mic19/CHCHD3, which is crucial for their stabilization in mammals. Importantly, we identified that the mitochondrial i-AAA protease Yme1L regulates Mic60/Mitofilin homeostasis. Impaired MICOS assembly causes the formation of 'giant mitochondria' because of dysregulated mitochondrial fusion and fission. Also, mtDNA nucleoids are disorganized and clustered in these giant mitochondria in which mtDNA transcription is attenuated because of remarkable downregulation of some key mtDNA nucleoid-associated proteins. Together, these findings demonstrate that Mic60/Mitofilin homeostasis regulated by Yme1L is central to the MICOS assembly, which is required for maintenance of mitochondrial morphology and organization of mtDNA nucleoids.
Project description:The MItochondrial Contact Site and Cristae Organizing System (MICOS) is required for the biogenesis and maintenance of mitochondrial cristae as well as the proper tethering of the mitochondrial inner and outer membranes. We recently demonstrated that the core components of MICOS, Mic10 and Mic60, are near-ubiquitous eukaryotic features inferred to have been present in the last eukaryote common ancestor. We also showed that Mic60 could be traced to α-proteobacteria, which suggests that mitochondrial cristae evolved from α-proteobacterial intracytoplasmic membranes. Here, we extend our evolutionary analysis to MICOS-interacting proteins (e.g., Sam50, Mia40, DNAJC11, DISC-1, QIL1, Aim24, and Cox17) and discuss the implications for both derived and ancestral functions of MICOS.
Project description:The conserved MICOS complex functions as a primary determinant of mitochondrial inner membrane structure. We address the organization and functional roles of MICOS and identify two independent MICOS subcomplexes: Mic27/Mic10/Mic12, whose assembly is dependent on respiratory complexes and the mitochondrial lipid cardiolipin, and Mic60/Mic19, which assembles independent of these factors. Our data suggest that MICOS subcomplexes independently localize to cristae junctions and are connected via Mic19, which functions to regulate subcomplex distribution, and thus, potentially also cristae junction copy number. MICOS subunits have non-redundant functions as the absence of both MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes. Mitochondrial defects resulting from MICOS loss are caused by misdistribution of respiratory complexes in the inner membrane. Together, our data are consistent with a model where MICOS, mitochondrial lipids and respiratory complexes coordinately build a functional and correctly shaped mitochondrial inner membrane.
Project description:The mitochondrial contact site and cristae organizing system (MICOS) is crucial for the formation of crista junctions and mitochondrial inner membrane architecture. MICOS contains two core components. Mic10 shows membrane-bending activity, whereas Mic60 (mitofilin) forms contact sites between inner and outer membranes. Here we report that Mic60 deforms liposomes into thin membrane tubules and thus displays membrane-shaping activity. We identify a membrane-binding site in the soluble intermembrane space-exposed part of Mic60. This membrane-binding site is formed by a predicted amphipathic helix between the conserved coiled-coil and mitofilin domains. The mitofilin domain negatively regulates the membrane-shaping activity of Mic60. Binding of Mic19 to the mitofilin domain modulates this activity. Membrane binding and shaping by the conserved Mic60-Mic19 complex is crucial for crista junction formation, mitochondrial membrane architecture and efficient respiratory activity. Mic60 thus plays a dual role by shaping inner membrane crista junctions and forming contact sites with the outer membrane.
Project description:The mitochondrial contact site and cristae organizing system (MICOS) is a multisubunit protein complex that is essential for the proper architecture of the mitochondrial inner membrane. MICOS plays a key role in establishing and maintaining crista junctions, tubular or slit-like structures that connect the cristae membrane with the inner boundary membrane, thereby ensuring a contiguous inner membrane. MICOS is enriched at crista junctions, but the detailed distribution of its subunits around crista junctions is unclear because such small length scales are inaccessible with established fluorescence microscopy. By targeting individually activated fluorophores with an excitation beam featuring a central zero-intensity point, the nanoscopy method called MINFLUX delivers single-digit nanometer-scale three-dimensional (3D) resolution and localization precision. We employed MINFLUX nanoscopy to investigate the submitochondrial localization of the core MICOS subunit Mic60 in relation to two other MICOS proteins, Mic10 and Mic19. We demonstrate that dual-color 3D MINFLUX nanoscopy is applicable to the imaging of organellar substructures, yielding a 3D localization precision of ?5 nm in human mitochondria. This isotropic precision facilitated the development of an analysis framework that assigns localization clouds to individual molecules, thus eliminating a source of bias when drawing quantitative conclusions from single-molecule localization microscopy data. MINFLUX recordings of Mic60 indicate ringlike arrangements of multiple molecules with a diameter of 40 to 50 nm, suggesting that Mic60 surrounds individual crista junctions. Statistical analysis of dual-color MINFLUX images demonstrates that Mic19 is generally in close proximity to Mic60, whereas the spatial coordination of Mic10 with Mic60 is less regular, suggesting structural heterogeneity of MICOS.
Project description:Remodeling of mitochondrial ultrastructure is a process that is critical for organelle physiology and apoptosis. Although the key players in this process-mitochondrial contact site and cristae junction organizing system (MICOS) and Optic Atrophy 1 (OPA1)-have been characterized, the mechanisms behind its regulation remain incompletely defined. Here, we found that in addition to its role in mitochondrial division, metallopeptidase OMA1 is required for the maintenance of intermembrane connectivity through dynamic association with MICOS. This association is independent of OPA1, mediated via the MICOS subunit MIC60, and is important for stability of MICOS and the intermembrane contacts. The OMA1-MICOS relay is required for optimal bioenergetic output and apoptosis. Loss of OMA1 affects these activities; remarkably it can be alleviated by MICOS-emulating intermembrane bridge. Thus, OMA1-dependent ultrastructure support is required for mitochondrial architecture and bioenergetics under basal and stress conditions, suggesting a previously unrecognized role for OMA1 in mitochondrial physiology.
Project description:Cristae architecture is important for the function of mitochondria, the organelles that play the central role in many cellular processes. The mitochondrial contact site and cristae organizing system (MICOS) together with the sorting and assembly machinery (SAM) forms the mitochondrial intermembrane space bridging complex (MIB), a large protein complex present in mammalian mitochondria that partakes in the formation and maintenance of cristae. We report here a new subunit of the mammalian MICOS/MIB complex, an armadillo repeat-containing protein 1 (ArmC1). ArmC1 localizes both to cytosol and mitochondria, where it associates with the outer mitochondrial membrane through its carboxy-terminus. ArmC1 interacts with other constituents of the MICOS/MIB complex and its amounts are reduced upon MICOS/MIB complex depletion. Mitochondria lacking ArmC1 do not show defects in cristae structure, respiration or protein content, but appear fragmented and with reduced motility. ArmC1 represents therefore a peripheral MICOS/MIB component that appears to play a role in mitochondrial distribution in the cell.
Project description: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 ?-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).