How to use isotope labeling and mass spectrometry to analyze the dynamic assembly of mitochondrial protein complexes

Johannes Herrmann – Frederik Sommer/Michael Schroda – Timo Mühlhaus – Bruce Morgan – Markus Räschle/Zuzana Storchova – Ralf Korn – Christoph Garth

Most cellular processes are carried out by multiprotein complexes. Already 15 years ago, 232 different protein complexes could be identified by mass spectrometry even in a simple organism such as yeast (Nature (2002) 415, 141f). The refinement of proteomic techniques allowed it to describe many more complexes since then, both in yeast and in other organisms, resulting in rather comprehensive lists of constituents that form the multiprotein assemblies of a cell. High-resolution structures exist for many of these complexes so that in many cases the positioning of each subunit is known. However, it still will be a major challenge to unravel the mechanistic processes by which complex protein structures are put together. For some specific model complexes, such as the 80S ribosome (Cell (2016) 166, 380f) or cytochrome c oxidase (Cell (2016) 167, 471f), the assembly process was studied in detail, leading to the identification of the different assembly steps and to many assembly factors and chaperones. However, for most cellular protein complexes, similar data are missing.

What we did over the previous three years

During the first BioComp period, we established methods to specifically enrich protein complexes and identify their composition (ref. 5), to analyze the composition of the mitochondrial proteome in different cell types (ref. 1&3) and to combine genetic manipulations with cellular fractionation for a profiling approach yielding in a comprehensive analysis of the protein composition of the mitochondrial ribosome (ref. 2&4). We currently develop a dynamic isotope labeling strategy to monitor the turnover of mitochondrial proteins in logarithmically growing yeast cultures.

What we plan for the next two years

We plan to combine the different strategies in order to follow the formation and turnover of entire protein complexes in living cells. To this end, we will design yeast strains in which mitochondrial transcription can be controlled. Upon de-repression, the formation of newly formed mitochondrial complexes will be followed using gradient centrifugation, dynamic isotope labeling and mass spectrometry. From our studies we expect fundamental insights into the mechanistic details by which mitochondrial protein complexes are assembled. In particular, we want to understand how fast these complexes assemble, in which order the components are put together, which helper proteins assist and control the assembly process and whether already assembled subunits stay within stable structures or whether they are dynamically exchanged among protein complexes.

Challenges in this project are concerning the experimental as well as the analytical part. This project requires the close cooperation within the framework of BioComp2 groups (see above). Our previous experience will be a solid basis for this demanding and exciting project.

References

[1] Ramesh A, Peleh V, Martinez-Caballero S, Wollweber F, Sommer F, van der Laan M, Schroda M, Alexander RT, Campo ML, Herrmann JM. 2016. A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import. J Cell Biol. 214, 417-31.

[2] Woellhaf MW, Sommer F, Schroda M, Herrmann JM. 2016. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein. Mol Biol Cell. 31. pii: mbc.E16-07-0513

[3] Peleh V, Cordat, E, Herrmann JM. 2016. Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding. eLIFE 25;5. pii: e16177

[4] Woellhaf MW, Hansen KG, Garth C, Herrmann JM. 2014. Import of ribosomal proteins into yeast mitochondria. Biochemistry and Cell Biology 92, 489-498

[5] Bode M, Woellhaf MW, Bohnert M, van der Laan M, Sommer F, Jung M, Zimmermann R, Schroda M, Herrmann JM. 2015. Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase. Mol Biol Cell 26, 2385-401