Import and assembly of mitochondrial outer membrane proteins - identification of new factors and import signals
Final Report Abstract
The project had two research focuses. One was to determine targeting signals that direct β-barrel proteins to mitochondria. Upon import, β-barrel proteins require the sorting and assembly machinery (the SAM complex) for integration into the outer mitochondrial membrane. To explore how βbarrel proteins are targeted to mitochondria, we wanted to make use of two highly similar porins, PorB from Neisseria gonorrhoeae and PorB from Neisseria mucosae, one of which was directed to mitochondria (PorBNgo), whereas the other one (PorBNmu) was not. We planned to exchange variable domains of these proteins and follow mitochondrial targeting of the chimerical constructs. However, initial experiments showed that PorBNmu was not expressed in human cells and all of the chimerical constructs we created were targeted to mitochondria. At the same time, we showed that Omp85Ngo, a neisserial homolog of Sam50, was imported into the human mitochondria and assembled into the outer membrane, where it could function in the membrane integration of neisserial PorB proteins. Enterobacterial homologs of Omp85Ngo (BamAEco) and PorBNgo (OmpCEco) were in spite of the similarity not imported into mitochondria. We decided, therefore, to exchange variable regions of these proteins. We could show that the carboxy-terminal part, and in the case of Omp85Ngo the β-sorting signal as well, play a role in the mitochondrial targeting of these proteins. In addition, we contributed to elucidation of the crystal structure of PorBNgo. The second focus of our research represented the SAM complex in the outer membrane of human mitochondria. Known components of the mammalian SAM complex are Sam50, Metaxin 1 and Metaxin 2. We created a knockdown cell line of Sam50 and, with the help of stable isotope labeling with amino acids in cell culture and quantitative mass spectrometry, we determined which other proteins are affected by Sam50 depletion. We hoped to find novel subunits of the SAM complex, but also to obtain a more generalized picture of what happens to mitochondria after such an important assembly factor as Sam50 is removed. We found out that Sam50 depletion strongly affects levels of various respiratory chain proteins, as well as TTC19 and C1orf163, two uncharacterized proteins. TTC19 has in the meantime been described to be a respiratory complex III assembly factor. Our work on C1orf163 showed that this is a novel respiratory complex IV assembly factor, which we termed Coa7. At the same time, we observed a connection between Sam50 depletion and loss of cristae structure. We were able to show that the SAM complex interacts strongly with the Mitofilin complex in the inner mitochondrial membrane, forming a mitochondrial intermembrane space bridging complex (MIB). The MIB complex plays a crucial role in cristae structuring and maintenance and is required for the proper assembly of respiratory chain complexes. In future, the many new perspectives our work opened will be followed, such as pinpointing the regions in PorBNgo required for its membrane integration and function, or further characterization of the MIB complex in human mitochondria and its connection to the respiratory chain assembly.
Publications
- (2011) Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria. J. Biol. Chem. 286: 27019-27026
Kozjak-Pavlovic, V., Ott, C., Gotz, M., Rudel, T.
- (2012) Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes. Mol. Cell. Biol. 32: 1173-1188
Ott, C., Ross, K., Straub, S., Thiede, B., Götz, M., Goosmann, C., Krischke, M., Mueller, M. J., Krohne, G., Rudel, T., Kozjak-Pavlovic, V.
(See online at https://doi.org/10.1128/MCB.06388-11) - (2012) Structure and function of the PorB porin from disseminating Neisseria gonorrhoeae. Biochem. J.
Zeth, K., Kozjak-Pavlovic, V., Faulstich, M., Fraunholz, M., Hurwitz, R., Kepp, O., Rudel, T.
(See online at https://doi.org/10.1042/BJ20121025)
