Analysis of the structure of the Oxa1-ribosome-complex by high-resolution cyro-electron microscopy

• Applicants: Professor Dr. Roland Beckmann; Professor Dr. Johannes M. Herrmann
• Subject Area: Biochemistry
• Term: from 2008 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 50070218

• Final Report Year: 2014

Final Report Abstract

Eukaryotic cells contain two distinct translation machineries, one in the cytosol and one in the mitochondrial matrix. Mitochondrial protein synthesis is specialized on the production of a small number of very hydrophobic membrane proteins. The necessity to insert these proteins into the inner membrane in a co-translational fashion presumably forced eukaryotic cell to make the effort to maintain a mitochondrial translation system. Owing to their specialization on the synthesis of membrane proteins, mitochondrial ribosomes (also mitoribosomes) are permanently tethered to the inner membrane. In this project we studied the structure and function of mitochondrial ribosomes with particular emphasis on the factors that facilitate protein insertion into the inner membrane. In the course of this project we could show that several membrane-bound factors associate with mitoribosomes in proximity to the polypeptide tunnel exit. These factors include the protein insertase Oxa1, the ribosome receptor Mba1, the inner membrane protein Mdm38, which cooperates with Mba1 in membrane binding, and Cbp3/Cbp6 which mediates cotranslational assembly of cytochrome b into cytochrome reductase. By single particle cryo-electron microscopy we could visualize the ribosome-binding domain of Oxa1 bound to the exit tunnel of the mitoribosome, and the entire membrane domain of the bacterial Oxa1 homolog YidC bound to an actively translating bacterial ribosome. Moreover, by cryo-electron tomography we could identify an rRNA extension loop as additional membrane contact of the large subunit of the fungal mitoribosome. Biochemical and genetic studies provided evidence that the close membrane contact of mitochondrial ribosomes is crucial both for co-translational protein insertion and for the subsequent assembly of the translation products, suggesting that the mitoribosome serves as a platform to coordinate early assembly stages of nascent chains. Our results demonstrated that the combination of structural and biochemical approaches are perfectly suited to unravel the mechanistic details of mitochondrial protein synthesis.

Publications

• 2010. Co-translational insertion of mitochondrially encoded proteins. Biochim Biophys Acta 1803, 767-775
  Ott M, Herrmann JM
  
• 2010. Cooperation of stop-transfer and conservative sorting mechanisms in mitochondrial protein transport. Curr Biol. 20, 1227-1232
  Bohnert M, Rehling P, Guiard B, Herrmann JM, Pfanner N, van der Laan M
  
• 2010. Proteins at the polypeptide tunnel exit of the yeast mitochondrial ribosome. J Biol Chem. 285, 19022-19028
  Gruschke S, Groene K, Heublein M, Hölz S, Israel L, Imhof A, Herrmann JM, Ott M
  
• 2010. Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation. Mol Biol Cell 21, 1937-1944
  Bauerschmitt H, Mick DU, Deckers M, Vollmer C, Funes S, Kehrein K, Ott M, Rehling P, Herrmann JM
  
• 2011. Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly. J Cell Biol. 193, 1101-1114
  Gruschke S, Kehrein K, Römpler K, Gröne K, Israel L, Imhof A, Herrmann JM, Ott M
  
• 2011. Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts. Biol Chem. 392, 13-19
  Funes S, Kauff F, van der Sluis EO, Ott M, Herrmann JM
  
• 2012. Control of protein synthesis in yeast mitochondria: The concept of translational activators. Biochim Biophys Acta. 1833, 286-294
  Herrmann JM, Woellhaf MW, Bonnefoy N
  
• 2012. Oxa1- ribosome complexes coordinate the assembly of cytochrome c oxidase in mitochondria. J Biol Chem. 287, 34484-34493
  Keil M, Bareth B, Woellhaf MW, Peleh V, Prestele M, Rehling P, Herrmann JM
  
• 2012. The Cbp3- Cbp6 complex coordinates cytochrome b synthesis with bc1 complex assembly in yeast mitochondria. J Cell Biol. 199, 137-150
  Gruschke S, Römpler K, Hildenbeutel M, Kehrein K, Kühl I, Bonnefoy N, Ott M
  
• 2012. The innermitochondrial distribution of Oxa1 depends on the growth conditions and on the availability of substrates. Mol Biol Cell 23, 2292-2301
  Stoldt S, Wenzel D, Hildenbeutel M, Wurm CA, Herrmann JM, Jakobs S
  
• 2012. The membrane insertase Oxa1 is required for efficient import of carrier proteins into mitochondria. J Mol Biol. 423, 590-599
  Hildenbeutel M, Theis M, Geier M, Haferkamp I, Neuhaus HE, Herrmann JM, Ott M
  
• 2012. The mitochondrial oxidase-assemblyprotein1 (Oxa1) insertase forms a membrane pore in lipid bilayers. J Biol Chem. 287, 33314-33326
  Krueger V, Deckers M, Hildenbeutel M, Van der Laan M, Hellmers M, Drekers C, Preuss M, Herrmann JM, Rehling P, Wagner R, Meinecke M
  
• 2014. A structural model of the active ribosome-bound membrane protein insertase YidC. eLife July 10
  Wickles S, Singharoy A, Andreani J, Seemayer S, Bischoff L, Berninghausen O, Soeding J, Schulten K, van der Sluis EO, Beckmann R
  
• 2014. Import of ribosomal proteins into yeast mitochondria. Biochem Cell Biol, 20, 1-10
  Woellhaf MW, Hansen KG, Garth C, Herrmann JM
  
• 2014. The Disulfide Relay of the Intermembrane Space Oxidizes the Ribosomal Subunit Mrp10 on Its Transit into the Mitochondrial Matrix. Developmental Cell 28, 30-42
  Longen S, Woellhaf MW, Petrungaro C, Riemer J, Herrmann JM