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Mitochondrial outer membrane β‐barrel proteins are encoded in the nucleus, translated in the cytosol and then targeted to and imported into the respective organelles. Detailed studies have uncovered the mechanisms involved in the import of these proteins and identified the targeting signals and the cytosolic factors that govern their proper biogenesis. Recently, de novo designed eight‐stranded β‐barrel proteins (Tmb2.3 and Tmb2.17) were shown to fold and assemble into lipid membranes. To better understand the general aspects of the biogenesis of β‐barrel proteins, we investigated the fate of these artificial proteins upon their expression in yeast cells. We demonstrate that although these proteins are de novo designed and are not related to bona fide mitochondrial β‐barrel proteins, they were targeted to mitochondria and integrated into the organelle outer membrane. We further studied whether this integration requires components of the yeast mitochondrial import machinery like Tom20, Tom70, Tob55/Sam50 and Mas37/Sam37. Whereas it seems that none of the import receptors was required for the biogenesis of the artificial β‐barrel proteins, we observed a strong dependency on the TOB/SAM complex. Collectively, our findings demonstrate that the mitochondrial outer membrane is the preferential location in yeast cells for any membrane‐embedded β‐barrel protein. [ABSTRACT FROM AUTHOR]

The respiratory chain in aerobic organisms is composed of a number of membrane-bound protein complexes that link electron transfer to proton translocation across the membrane. In mitochondria, the final electron acceptor, complex IV (CIV), receives electrons from dimeric complex III (CIII2), via a mobile electron carrier, cytochrome c. In the present study, we isolated the CIII2CIV supercomplex from the fission yeast Schizosaccharomyces pombe and determined its structure with bound cyt. c using single-particle electron cryomicroscopy. A respiratory supercomplex factor 2 was found to be bound at CIV distally positioned in the supercomplex. In addition to the redox-active metal sites, we found a metal ion, presumably Zn2+, coordinated in the CIII subunit Cor1, which is encoded by the same gene (qcr1) as the mitochondrial-processing peptidase subunit ß. Our data show that the isolated CIII2CIV supercomplex displays proteolytic activity suggesting a dual role of CIII2 in S. pombe. As in the supercomplex from S. cerevisiae, subunit Cox5 of CIV faces towards one CIII monomer, but in S. pombe, the two complexes are rotated relative to each other by ~45°. This orientation yields equal distances between the cyt. c binding sites at CIV and at each of the two CIII monomers. The structure shows cyt. c bound at four positions, but only along one of the two symmetrical branches. Overall, this combined structural and functional study reveals the integration of peptidase activity with the CIII2 respiratory system and indicates a two-dimensional cyt. c diffusion mechanism within the CIII2-CIV supercomplex. [ABSTRACT FROM AUTHOR]

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts harbor [beta]-barrel proteins. The signals that allow precursors of such proteins to be targeted to mitochondria were not characterized so far. To better understand the mechanism by which [beta]-barrel precursor proteins are recognized and sorted within eukaryotic cells, we expressed the bacterial [beta]-barrel proteins PhoE, OmpA, Omp85, and OmpC in Saccharomyces cerevisiae and demonstrated that they were imported into mitochondria. A detailed investigation of the import pathway of PhoE revealed that it is shared with mitochondrial [beta]-barrel proteins. PhoE interacts initially with surface import receptors, and its further sorting depends on components of the TOB/SAM complex. The bacterial Omp85 and PhoE integrated into the mitochondrial outer membrane as native-like oligomers. For the latter protein this assembly depended on the C-terminal Phe residue, which is important also for the correct assembly of PhoE into the bacterial outer membrane. Collectively, it appears that mitochondrial [beta]-barrel proteins have not evolved eukaryotic-specific signals to ensure their import into mitochondria. Furthermore, the signal for assembly of [beta]-barrel proteins into the bacterial outer membrane is functional in mitochondria. outer membrane | PhoE | protein import | TOB complex | TOM complex

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import [beta]-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by ~4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly [beta]-barrel proteins.

[beta]-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of [beta]-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of [beta]-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both [barrel]-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane [beta]-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial [beta]-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.

Author(s): Stefan A. Paschen [1]; Thomas Waizenegger [1]; Tincuta Stan [1]; Marc Preuss [1]; Marek Cyrklaff [2]; Kai Hell [1, 3]; Doron Rapaport [1, 3]; Walter Neupert (corresponding author) [1] [...]

The translocase at the outer membrane of mitochondria (TOM complex) mediates the initial steps of the import of preproteins into the organelle, which are essential for mitochondrial biogenesis and, therefore, for eukaryotic cell viability. The TOM complex is a multisubunit molecular machine with a dynamic structure. The biogenesis of TOM is of special interest because the complex is required for its own biogenesis. This article describes the mechanisms by which Tom components are targeted to the mitochondria and inserted into the outer membrane. The assembly of newly synthesized subunits into the functional TOM complex might occur via assembly intermediates that are in equilibrium with the mature complex.