f 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).
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.
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.
As a consequence of their bacterial origin, mitochondria contain -barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the -barrel assembly machinery (BAM) required for biogenesis of -barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial -barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only -barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial -barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of -barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of -barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.Mitochondria are organelles of bacterial origin, surrounded by an outer (OMM) 4 and an inner membrane (IMM). The OMM contains -barrel proteins, a class of pore-forming proteins additionally found only in chloroplasts and Gram-negative bacteria (1, 2). Similar to the majority of other mitochondrial proteins, -barrel proteins are synthesized in the cytosol and have to be imported into mitochondria with the help of the translocase of the outer mitochondrial membrane (TOM) complex. For the membrane integration and assembly into complexes, -barrel proteins require additional proteinaceous machinery in the OMM. This is so called sorting and assembly machinery (SAM), also known as topogenesis of outer membrane -barrel proteins (TOB complex) (3, 4).The central component of the SAM complex is Sam50/ Tob55, a protein with a function that has been conserved from bacteria to human (4 -6). Other components of the SAM complex include the Sam35/Tob38/Tom38 (7-9) and Sam37/ Mas37/Tom37 (3, 10) proteins in yeast, and Metaxin 1 and Metaxin 2 (11) in mammalian mitochondria.The signals in -barrel proteins that are recognized by the TOM complex belong to internal targeting signals and are not yet fully understood. However, a specific signal has been identified in the C-terminal part of mitochondrial -barrel proteins that directs them to the SAM complex to be properly sorted and integrated into the OMM (12). Unlike in yeast, this signal in mammalian -barrel proteins is always present at the extreme C terminus of the protein, and the addition of even a shor...
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