Marcos Contextuales

The vast majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol as precursor proteins and subsequently transported into mitochondria. During this process, essentially all precursor proteins of the different subcompartments cross the mitochondrial outer membrane through the TOM complex. The TOM complex is made up by a protein conducting channel that is formed by the β-barrel protein Tom40, the receptors Tom20, Tom22 and Tom70 and the small subunits Tom5, Tom6 and Tom7. It is quite well understood how precursor proteins are recognized by the receptors, pass through the TOM complex and reach their final destination. However, it is not known whether the TOM pore can also open laterally to mediate insertion of precursor proteins into the outer membrane.

In the present study GFP-Tim23 fusion proteins were used to analyze this problem. In this artificial protein GFP is fused to the N-terminus of Tim23. The wild type form of Tim23 is integrated with four C-terminally located hydrophobic segments into the inner membrane whereas the N-terminal part is located in the intermembrane space. Depending on the protein import activity of the TIM23 translocase the N-terminal 20 amino acid residues of Tim23 can be exposed on the sucface of the outer membrane. An interesting behavior of GFP-Tim23 upon import was shown before; GFP-Tim23 passes the TOM pore, the Tim23 domain is integrated in the inner membrane but the GFP domain remains on the outer surface of the mitochondria facing the cytosol. This results in two possible locations: The protein gets either stuck in the channel of the TOM complex or it is released into the outer membrane. It is shown here, that the topology of GFP-Tim23 can be altered by changing the growth temperature. Upon growth on 24°C the GFP domain is present on the surface of the outer membrane whereas it is located in the intermembrane space when the yeast strain expressing this fusion protein is grown on 37°C. Furthermore, it is shown in this study, that GFP-Tim23 can indeed be released laterally from the TOM channel when the GFP domain is facing the cytosol. The release is dependent on sequence determinants present in the N-terminal segment of the Tim23 domain of the fusion protein. As a result the GFP-Tim23 fusion protein spans both, the inner and the outer mitochondrial membrane. Therefore, the TOM complex can act not only as a protein translocase but also as an insertase that mediates lateral release into the outer membrane. This represents a new topogenesis pathway of proteins in mitochondria.

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The experiments were also performed to be able to address another question. A tool should be generated to analyze the molecular basis of contact sites between the outer membrane and the inner boundary membrane of mitochondria. Mitochondria are surrounded by an envelope consisting of these two membranes. At limited sites, called contact sites, these two membranes are firmly connected. Although the presence of these structures is known since a long time, their molecular nature remained unknown. Since GFP-Tim23 is integrated in both mitochondrial membranes, it can be used as a marker for these structures.

By using the double membrane spanning GFP-Tim23 as a marker protein, in this study a protocol was designed to generate membrane vesicles from yeast mitochondria and separate them in such a way that a fraction is obtained, in which contact site proteins are specifically enriched. Based on these results six proteins could be identified showing an almost identical distribution as GFP-Tim23 and thus fulfilling the criteria for contact site proteins. The identification was achieved by quantitative high-resolution mass spectrometry using the SILAC approach (Stable Isotope Labeling with Amino acids in Cell culture). The six proteins identified are Fcj1, the yeast mitofilin, and five uncharacterized membrane integral or associated proteins which were named Mcs10, Mcs12, Mcs19, Mcs27 and Mcs29. Mcs stands for “mitochondrial contact sites”. They all interact with each other and form a novel high molecular mass complex which was termed MICOS complex (mitochondrial contact site complex). Furthermore, a search was performed for interaction partners of MICOS in the outer membrane. Two protein complexes were identified which form contacts with MICOS, the TOB complex and the Ugo1-Fzo1 complex. Their, so far known, functions are the insertion of β-barrel proteins into the mitochondrial outer membrane and the fusion of the outer membranes. These interactions apparently represent additional functions of these complexes of the outer membrane. Immunogold labeling showed that the MICOS complex is located preferentially at the sites where the inner boundary membrane meets the crista membrane, the crista junctions. Deletion of components of the MICOS complex in some cases leads to a virtually complete loss or in other cases to a reduction of crista junctions. This indicates an essential role of the MICOS complex in the formation and/or the stability of crista junctions. In addition the disturbance of the mitochondrial architecture results in a reduction of respiratory competence or the virtually complete loss of it. These results demonstrate an intimate connection between architecture and function of mitochondria.

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