Logo Logo
Switch language to English
Habib, Shukry James (2006): Biogenesis and function of mitochondrial outer membrane proteins. Dissertation, LMU München: Fakultät für Biologie



The mitochondrial outer membrane harbors different sub-classes of proteins that mediate numerous interactions between the mitochondrial metabolic and genetic system and the rest of the eukaryotic cell. Two classes of these proteins were investigated in this thesis. The first class, tail-anchored proteins, exposes a large domain to the cytosol and is anchored to the membrane by a single hydrophobic segment close to the C-terminus. This segment is usually flanked by positively–charged amino acids residues. In the first part of my study I could identify that the tail anchor domain fulfills four distinct functions: First, the tail anchor domain mediates the targeting to mitochondria in a process that probably requires the positive charges down stream the transmembrane segment. Second, the domain facilitates the insertion into the outer membrane. Third, the tail anchor domain is required for assembly of the proteins into the relevant complexes. Finally, it can stabilize such complexes. In the second part of my study I investigated the biogenesis of β-barrel proteins. These membrane proteins are unique for the outer membrane of mitochondria, chloroplast and gram-negative bacteria. Recently a complex which mediates the biogenesis of β-barrel proteins was identified and termed TOB (SAM) complex. At the beginning of this work, the TOB complex was known to consist of the peripheral associated membrane protein Mas37 and the essential membrane embedded component Tob55. Initially, we could identify Tob38 as a new component of the TOB complex. Tob38 is encoded by an essential gene and the protein associates with the TOB complex at the cytosolic side of mitochondria. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38–Tob55 core complex binds precursors of β-barrel proteins and facilitates their insertion into the outer membrane. The import of β-barrel precursors into Tob38-depleted mitochondria was demonstrated here to be dramatically reduced in comparison to wild type organelles. Notably, such an effect was not observed for other precursors of the outer membrane proteins and precursors distained to the various sub-mitochondrial compartments. Taken together, we conclude that Tob38 has a crucial function in the biogenesis of β-barrel proteins of mitochondria. Next, the import and the assembly pathways of Mas37 and Tob55 were analyzed in detail. Reduced insertion of the Tob55 precursor in the absence of Tom20 and Tom70 argues for initial recognition of the precursor of Tob55 by the import receptors. It is then transferred through the import channel formed by Tom40. Variants of the latter protein influenced the insertion of Tob55. Assembly of newly synthesized Tob55 into pre-existing TOB complexes, as analyzed by blue native gel electrophoresis, depended on pre-existing Tob55 and Tob38 but to a less extent on Mas37. In contrast, both the association of Mas37 precursor with mitochondria and its assembly into the TOB complex were not affected by mutation in the TOM complex. My results suggest that Mas37 assembled directly with the TOB core complex. Hence, the biogenesis of Mas37 represents a novel import pathway of mitochondrial proteins. Finally, the structure function relationships of Tob55 were investigated by combination of biochemical and genetic approaches. Tob55 exposes an N-terminal hydrophilic domain into the intermembrane space and is anchored in the outer membrane by its C-terminal β-barrel domain. Deletion of various lengths at the N-terminal domain did not affect the targeting of Tob55 precursor to mitochondria and its insertion into the outer membrane. Replacement of wild type Tob55 by these deletion variants resulted in reduced growth of cells. Mitochondria isolated from such cells contain reduced levels of β-barrel proteins and are impaired in their capacity to import newly synthesized β-barrel precursors. Finally, purified N-terminal domain of Tob55 was found to be able to bind β-barrel precursors in a specific manner. Taken together, these results demonstrate that the N-terminal domain of Tob55 has a function in recognizing precursors of β-barrel proteins. Furthermore, the receptor-like function of the N-terminal domain of Tob55 seems to have a role in coupling the translocation of β-barrel precursors across the TOM complex to their interaction with the TOB complex.