Protein import into the intermembrane space of mitochondria
Final Report Abstract
Mitochondria are organelles essential for the life of eukaryotic cells. Proteins of the intermembrane space (IMS), which is delimited by outer and inner membrane, are involved in many crucial cellular and biochemical process, including energy production by oxidative phosphorylation, assembly of respiratory chain complexes, transport of biomolecules and apoptotic signalling. Interestingly, all IMS proteins are synthesized in the cytosol. Subsequently, they have to be imported across the outer membrane into the IMS. The redox receptor Mia40 and the thiol oxidase Erv1 form a Mia40/Erv1-disulfide relay system (DRS) that introduces disulfide bonds in classical twin CxnC-motif proteins thereby driving their import into the IMS. In addition, the DRS has non-classical substrates harbouring disulphide bonds. Despite the importance of the Mia40/Erv1 import pathway, the early steps of this import pathway in the cytosol and the import of non-classical IMS proteins harbouring disulphide bonds remain mostly elusive. We hypothesised that cytosolic proteins are required to keep the precursors of Mia40 substrates in a reduced import competent state in the cytosol. In the first project, we aimed to identify cytosolic proteins interacting with Mia40 substrates. We accumulated tagged Mia40 substrate in the cytosol, followed by their isolation and subsequent mass spectrometry of coisolated proteins. Using this approach, we co-purified members of the Hsp70 chaperone superfamily with cytosolic Mia40 substrates, e.g. the Ssa proteins and the Sse proteins. In absence of functional Ssa proteins, we observed a defect in the biogenesis of Mia40 substrates. In a second project, we were interested in the location and biogenesis of Mix17. We clearly demonstrated that Mix17 is exposed to the cytosol. Unlike all other Mia40 substrates, Mix17 spans the OM with an Nout-Cin-topology. The insertion of Mix17 into the OM depends on its N terminus, the energetic state of mitochondria and most likely the TOM complex. In summary, we identified Mix17 as a novel kind of Mia40 substrate. By providing important information about the location and biogenesis of Mix17, this study generates the basis for elucidating the function of Mix17 and its orthologs in higher eukaryotes in the future. In the third project, we characterised molecular defects caused by patients’ derived GFER mutations in the yeast system. In agreement with their growth phenotype in yeast, the newly identified mutations showed modest effects on the redox state of Mia40 and the efficiency of the Mia40 import pathway. Strikingly, growing the mutant strains in hypoxia significantly enhanced their growth defect. These results provide first insights in the molecular basis of the disease phenotype caused by the mutations.
Publications
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A single-cysteine mutant and chimeras of essential Leishmania Erv can complement the loss of Erv1 but not of Mia40 in yeast. Redox Biology, 15, 363-374.
Specht, Sandra; Liedgens, Linda; Duarte, Margarida; Stiegler, Alexandra; Wirth, Ulrike; Eberhardt, Maike; Tomás, Ana; Hell, Kai & Deponte, Marcel
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The Mia40 substrate Mix17 exposes its N-terminus to the cytosolic side of the mitochondrial outer membrane. Cold Spring Harbor Laboratory.
Resch, Moritz; Frickel, Johanna S.; Dischinger, Korbinian; Wen, Rachel Choo Shen; Hell, Kai & Harner, Max E.
